U.S. patent application number 14/227759 was filed with the patent office on 2014-10-02 for method of combination therapy using an egfr antagonist and anti-c-met antibody.
This patent application is currently assigned to SAMSUNG LIFE WELFARE FOUNDATION. The applicant listed for this patent is SAMSUNG ELECTRONICS CO., LTD., SAMSUNG LIFE WELFARE FOUNDATION. Invention is credited to Jae Hyun Choi, Yun Ju Jeong, Kyung Ah Kim, Ji Min Lee, Saet Byoul LEE.
Application Number | 20140294830 14/227759 |
Document ID | / |
Family ID | 51621081 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140294830 |
Kind Code |
A1 |
LEE; Saet Byoul ; et
al. |
October 2, 2014 |
METHOD OF COMBINATION THERAPY USING AN EGFR ANTAGONIST AND
ANTI-C-MET ANTIBODY
Abstract
A method of combination therapy for prevention and/or treatment
of c-Met- and/or EGFR-induced diseases including co-administering a
pharmaceutically effective amount of an EGFR antagonist and a
pharmaceutically effective amount of an anti-c-Met antibody to a
subject in need thereof is provided.
Inventors: |
LEE; Saet Byoul; (Seoul,
KR) ; Choi; Jae Hyun; (Seongnam-si, KR) ; Kim;
Kyung Ah; (Seongnam-si, KR) ; Lee; Ji Min;
(Seoul, KR) ; Jeong; Yun Ju; (Hwaseong-si,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAMSUNG LIFE WELFARE FOUNDATION
SAMSUNG ELECTRONICS CO., LTD. |
Seoul
Suwon-si |
|
KR
KR |
|
|
Assignee: |
SAMSUNG LIFE WELFARE
FOUNDATION
Seoul
KR
SAMSUNG ELECTRONICS CO., LTD.
Suwon-si
KR
|
Family ID: |
51621081 |
Appl. No.: |
14/227759 |
Filed: |
March 27, 2014 |
Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/136.1; 424/139.1 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61K 39/3955 20130101; A61K 31/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 31/517 20130101; A61K 31/517 20130101;
A61K 39/3955 20130101; A61K 31/5377 20130101; A61P 35/00
20180101 |
Class at
Publication: |
424/135.1 ;
424/139.1; 424/133.1; 424/136.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/5377 20060101 A61K031/5377; A61K 31/517
20060101 A61K031/517 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2013 |
KR |
10-2013-0032842 |
Claims
1. A method for prevention or treatment of a c-Met-induced or
EGFR-induced disease, comprising co-administering (a) an EGFR
antagonist and (b) an anti-c-Met antibody or antigen-binding
fragment thereof to a subject in need thereof, wherein the
anti-c-Met antibody or the antigen-binding fragment thereof
specifically binds to an epitope comprising 5 or more contiguous
amino acids within the SEMA domain of c-Met protein.
2. The method of claim 1, wherein the EGFR antagonist and the
anti-c-Met antibody or antigen-binding fragment thereof are
administered simultaneously or sequentially in any order.
3. The method of claim 1, wherein the EGFR antagonist comprises at
least one selected from the group consisting of a small-molecule
tyrosine inhibitor and an anti-EGFR antibody.
4. The method of claim 3, wherein the EGFR antagonist comprises at
least one selected from the group consisting of erlotinib,
gefitinib, cetuximab, and panitumumab.
5. The method of claim 1, wherein the anti c-Met antibody or the
antigen-binding fragment thereof specifically binds to an epitope
comprising 5 to 19 contiguous amino acids of SEQ ID NO: 71, and
wherein the epitope comprises the amino acid sequence of SEQ ID NO:
73.
6. The method of claim 1, wherein the anti c-Met antibody or the
antigen-binding fragment thereof comprises: a heavy chain variable
region comprising at least one heavy chain complementarity
determining region (CDR) selected from the group consisting of (a)
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID
NO: 2, or an amino acid sequence comprising 8-19 consecutive amino
acids of SEQ ID NO: 2 comprising the amino acid residues from the
3.sup.rd to 10.sup.th positions of SEQ ID NO: 2; and (c) a CDR-H3
comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85,
or an amino acid sequence comprising 6-13 consecutive amino acids
of SEQ ID NO: 85 comprising the amino acid residues from the
1.sup.st to 6.sup.th positions of SEQ ID NO: 85; and a light chain
variable region comprising at least one light chain complementarity
determining region (CDR) selected from the group consisting of (a)
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c)
a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID
NO: 86, or an amino acid sequence comprising 9-17 consecutive amino
acids of SEQ ID NO: 89 comprising the amino acid residues from the
1.sup.st to 9.sup.th positions of SEQ ID NO: 89.
7. The method of claim 6, wherein the CDR-H1 has the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID
NO: 24, the CDR-H2 has the amino acid sequence of SEQ ID NO: 2, SEQ
ID NO: 25, or SEQ ID NO: 26, the CDR-H3 has the amino acid sequence
of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 85,
the CDR-L1 has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:
29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or
SEQ ID NO: 106, the CDR-L2 has the amino acid sequence of SEQ ID
NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and the
CDR-L3 has the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID
NO: 86, or SEQ ID NO: 89.
8. The method of claim 6, wherein the heavy chain variable region
has the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 74, SEQ ID
NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93,
or SEQ ID NO: 94, and the light chain variable region has the amino
acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ
ID NO: 21, SEQ ID NO: 75, SEQ ID NO: 88, SEQ ID NO: 95, SEQ ID NO:
96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO:
107.
9. The method of claim 6, wherein the anti-c-Met antibody
comprises: a heavy chain comprising the amino acid sequence of SEQ
ID NO: 62, the amino acid sequence from the 18.sup.th to 462.sup.nd
positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO:
64, the amino acid sequence from the 18.sup.th to 461.sup.st
positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO:
66, or the amino acid sequence from the 18.sup.th to 460.sup.th
positions of SEQ ID NO: 66; and a light chain comprising the amino
acid sequence of SEQ ID NO: 68, the amino acid sequence from the
21.sup.st to 240.sup.th positions of SEQ ID NO: 68, the amino acid
sequence of SEQ ID NO: 70, the amino acid sequence from the
21.sup.st to 240.sup.th positions of SEQ ID NO: 70, or the amino
acid sequence of SEQ ID NO: 108.
10. The method of claim 1, wherein the anti-c-Met antibody is a
monoclonal antibody.
11. The method of claim 1, wherein the anti-c-Met antibody is a
mouse antibody, a mouse-human chimeric antibody, a humanized
antibody, or a human antibody.
12. The method of claim 1, wherein the antigen-binding fragment is
selected from the group consisting of scFv, (scFv).sub.2, Fab,
Fab', and F(ab').sub.2 of the anti-c-Met antibody.
13. The method of claim 1, wherein the anti-c-Met antibody is
provided by a bispecific antibody comprising an anti-c-Met antibody
or an antigen-binding fragment thereof and a VEGF-binding
fragment.
14. The method of claim 13, wherein the VEGF-binding fragment is an
anti-VEGF antibody, an antigen-binding fragment of the anti-VEGF
antibody, a VEGF receptor, or a VEGF-binding region of the VEGF
receptor.
15. The method of claim 13, wherein the VEGF-binding fragment is
bevacizumab, an antigen-binding fragment of bevacizumab, human VEGF
Receptor 1 (SEQ ID NO: 113), the second Ig-like domain 2 (VIG2) of
SEQ ID NO: 114, or a polypeptide comprising 101 to 1338 consecutive
amino acids within the amino acid sequence of SEQ ID NO: 113,
wherein the 101 to 1338 consecutive amino acids comprises SEQ ID
NO: 114.
16. The method according to claim 13, wherein the bispecific
antibody further comprises a linker comprising 1 to 100 amino acids
between the anti-c-Met antibody or the antigen-binding fragment
thereof and the VEGF-binding fragment.
17. The method according to claim 1, wherein the c-Met-induced or
EGFR-induced disease is cancer or autosomal dominant polycystic
kidney disease (ADPKD).
18. A method for overcoming resistance to an EGFR antagonist,
comprising administering an EGFR antagonist together with an
anti-c-Met antibody or antigen-binding fragment thereof to a
subject in need thereof, wherein the anti-c-Met antibody or the
antigen-binding fragment thereof specifically binds to an epitope
comprising 5 or more contiguous amino acids within the SEMA domain
of c-Met protein.
19. The method of claim 18, wherein the anti c-Met antibody or the
antigen-bind ing fragment thereof comprises: a heavy chain variable
region comprising at least one heavy chain complementarity
determining region (CDR) selected from the group consisting of (a)
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID
NO: 2, or an amino acid sequence comprising 8-19 consecutive amino
acids of SEQ ID NO: 2 comprising amino acid residues from the
3.sup.rd to 10.sup.th positions of SEQ ID NO: 2; and (c) a CDR-H3
comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85,
or an amino acid sequence comprising 6-13 consecutive amino acids
of SEQ ID NO: 85 comprising amino acid residues from the 1.sup.st
to 6.sup.th positions of SEQ ID NO: 85; and a light chain variable
region comprising at least one light chain complementarity
determining region (CDR) selected from the group consisting of (a)
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c)
a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID
NO: 86, or an amino acid sequence comprising 9-17 consecutive amino
acids of SEQ ID NO: 86 comprising amino acid residues from the
1.sup.st to 9.sup.th positions of SEQ ID NO: 89.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of Korean Patent
Application No. 10-2013-0032842 filed on Mar. 27, 2013 in the
Korean Intellectual Property Office, the entire disclosure of which
is herein incorporated by reference.
INCORPORATION-BY-REFERENCE MATERIAL ELECTRONICALLY SUBMITTED
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
herewith and identified as follows: 147,384 bytes ASCII (Text) file
named "714092_ST25.TXT," created Mar. 24, 2014.
BACKGROUND
[0003] 1. Field
[0004] Provided is a method of combination therapy for prevention
and/or treatment of c-Met- and/or EGFR-induced diseases including
co-administering a pharmaceutically effective amount of an EGFR
antagonist and a pharmaceutically effective amount of an anti-c-Met
antibody to a patient in need thereof.
[0005] 2. Description of the Related Art
[0006] c-Met, a typical receptor tyrosine kinase (RTK) present at
the surface of cells, binds to its ligand, hepatocyte growth factor
(HGF) to promote intracellular signal transduction, thereby not
only promoting the growth of cells but also being over-expressed in
cancer cells so that it is widely implicated in cancer incidence,
cancer metastasis, cancer cell migration, cancer cell penetration,
angiogenesis, etc.
[0007] In general, common cancer therapies include surgery,
chemotherapy, radiotherapy, or a combination thereof. These
therapies have significant limits. For example, surgery may not
completely remove newly-generated tissues, and may not be used for
treatment of several disseminated neoplastic conditions such as
acute lymphoblastic leukemia. Radiotherapy may be effective only
when neoplastic tissues exhibit higher sensitivity to radiation
than normal tissues, and may cause serious side effects.
[0008] Although various anticancer drugs may be used in
chemotherapy, almost all anticancer drugs are toxic and frequently
cause dangerous side effects. In addition, many tumor cells exhibit
resistance to various anticancer drugs used in chemotherapy, which
becomes a serious obstacle in anticancer therapy.
[0009] To counteract the side effects and decrease resistance,
studies relating to combined use of two or more anticancer drugs
are being actively progressed. Since a combination therapy may
achieve synergistic effects of two or more anticancer drugs,
combination therapies are becoming a main trend in recent
anticancer therapies.
[0010] Accordingly, there is a demand for development of a
combination therapy for achieving increased synergistic effects and
decreased resistance.
SUMMARY
[0011] Applicant has discovered that the combination therapy of an
anti-c-Met antibody and an epidermal growth factor receptor (EGFR)
antagonist could achieve significant synergistic effects even if
EGFR antagonist resistance occurs.
[0012] Accordingly, one embodiment of the invention provides a
method of combination therapy for prevention and/or treatment of
c-Met and/or EGFR induced diseases, including co-administering a
pharmaceutically effective amount of an EGFR antagonist and a
pharmaceutically effective amount of an anti-c-Met antibody to a
subject in need of prevention and/or treatment of c-Met and/or EGFR
induced diseases.
[0013] Another embodiment provides a pharmaceutical composition for
combination therapy for prevention and/or treatment of c-Met and/or
EGFR induced diseases, including pharmaceutically effective amounts
of an EGFR antagonist and an anti-c-Met antibody as active
ingredients.
[0014] Still another embodiment provides a kit for combination
therapy for prevention and/or treatment of c-Met and EGFR induced
diseases, inducing a first pharmaceutical composition including a
pharmaceutically effective amount of an EGFR antagonist as an
active ingredient, a second pharmaceutical composition including a
pharmaceutically effective amount of an anti-c-Met antibody as an
active ingredient, and a package container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a graph showing the cell growth level of HCC827
cell lines treated with erlotinib and anti-c-Met antibody L3-1Y
alone or in combination.
[0016] FIG. 2 is a graph showing the level of c-Met genes of HCC827
erlotinib resistant (ER) cell lines measured by real-time PCR.
[0017] FIG. 3 is a graph showing the level of EGFR antagonist
resistance of HCC827 erlotinib resistant cell line.
[0018] FIGS. 4A-4D are graphs showing the profiles of cell growth
of HCC827 erlotinib resistant cell lines treated with erlotinib and
various anti-c-Met antibodies alone or in combination.
[0019] FIG. 5 is a graph showing the cell viability of HCC827
erlotinib resistant cell line #15 treated with erlotinib and
various anti-c-Met antibodies in combination.
[0020] FIG. 6 is an image showing the profile of expression of
c-Met, EGFR, c-Cbl, and LRIG1 in HCC827 erlotinib resistant cell
lines #10 and #15, respectively.
[0021] FIG. 7 is a schematic that shows the structure of a
bispecific antibody including an anti-c-Met antibody and a
VEGFR1-IG2 domain which are coupled to each other.
DETAILED DESCRIPTION
[0022] The present invention relates to a co-administration of an
anti-c-Met antibody that induces the degradation of c-Met and an
EGFR antagonist. More specifically, the present invention provides
a method of a combination therapy with excellent synergistic
effects and excellent anticancer effects for various kinds of
cancer cells, particularly EGFR antagonist resistant cancer, by
simultaneously inhibiting the actions of HGF/c-Met and HER1/EGFR,
which are known to be important growth factors of cancer cells.
[0023] In various anticancer chemotherapies targeting EGFR, if drug
resistance occurs, EGFR-targeting treatment may not achieve effects
any longer. In this case, if an EGFR antagonist and an anti-c-Met
antibody are co-administered, significant synergistic effects may
be achieved compared to the administration of only an EFGR
antagonist or an anti-c-Met antibody, and resistance to the EGFR
antagonist may be overcome, to achieve the original therapeutic
effects.
[0024] In anticancer chemotherapy, resistance to an anti-cancer
agent may further aggravate cancer, and continuously cause
resistance to other drugs, and finally, there may be no applicable
drugs thus rendering the cancer treatment impossible. Thus,
overcoming resistance to an anticancer agent is very important in
anticancer chemotherapy.
[0025] In the case of EGFR antagonist resistant lung cancer, it is
known that about 20% of patients have an amplified c-Met expression
level, and thus, it appears that the amplification of c-Met is
related to a mechanism whereby EGFR antagonist resistance occurs.
Thus, a therapy for EGFR antagonist resistant cancers by
simultaneously inhibiting EGFR and c-Met may be a solution to
overcome the resistance.
[0026] However, if some of the existing anti-c-Met antibody is
co-administered with an EGFR antagonist for EGFR antagonist
resistant cancer, synergistic effects and resistance overcoming
effects may not be achieved or may be insignificant, while it was
confirmed that if an EGFR antagonist and an anti-c-Met antibody as
defined below are co-administered, significant anticancer
synergistic effects and resistance overcoming effects may be
achieved. Thus, the present invention presents a therapy
particularly useful for a patient with resistance to an anticancer
agent targeting EGFR.
[0027] Accordingly, one embodiment provides a pharmaceutical
composition for combination therapy (co-administration) for
prevention and/or treatment of a c-Met- and/or EGFR-induced
disease, comprising an EGFR antagonist and an anti-c-Met antibody
as active ingredients. Another embodiment provides a method for
prevention and/or treatment of a c-Met-induced and/or EGFR-induced
disease, including co-administering an EGFR antagonist and an
anti-c-Met antibody or antigen-binding fragment thereof to a
subject in need thereof.
[0028] Another embodiment provides a pharmaceutical composition for
combination therapy (co-administration) for overcoming resistance
to an EGFR antagonist, including an EGFR antagonist and an
anti-c-Met antibody as active ingredients. Another embodiment
provides a method for overcoming resistance to an EGFR antagonist,
including administering an EGFR antagonist together with an
anti-c-Met antibody or antigen-binding fragment thereof to a
subject in need thereof.
[0029] In one particular embodiment, the pharmaceutical composition
for combination therapy may be formulated as a mixed formulation by
mixing a pharmaceutically effective amount of an EGFR antagonist
and a pharmaceutically effective amount of an anti-c-Met antibody
for co-administration, to be simultaneously administered as a
combined mixture.
[0030] In another particular embodiment, the pharmaceutical
composition for combined therapy may be one where a
pharmaceutically effective amount of an EGFR antagonist and a
pharmaceutically effective amount of an anti-c-Met antibody are
formulated, respectively, and are then administered simultaneously
or sequentially. The pharmaceutical composition for combined
therapy may be a pharmaceutical composition for simultaneous or
sequential administration, including a first pharmaceutical
composition including a pharmaceutically effective amount of an
EGFR antagonist as an active ingredient and a second pharmaceutical
composition including a pharmaceutically effective amount of an
anti-c-Met antibody as an active ingredient. For sequential
administration, the sequence of administration (which active
ingredient is administered first or second) may be in any
order.
[0031] Another embodiment provides a kit for prevention and/or
treatment of c-Met and EGFR induced diseases, including (a) a first
pharmaceutical composition containing a pharmaceutically effective
amount of an EGFR antagonist as an active ingredient, (b) a second
pharmaceutical composition containing a pharmaceutically effective
amount of anti-c-Met antibody as an active ingredient, and (c) a
package container.
[0032] Another embodiment provides a method of combination therapy
for prevention and/or treatment of a c-Met- and/or EGFR-induced
disease, including co-administering a pharmaceutically effective
amount of an EGFR antagonist and a pharmaceutically effective
amount of anti-c-Met antibody to a subject in need of prevention
and/or treatment of the c-Met- and/or EGFR-induced disease. In one
particular embodiment, the co-administration may be conducted by
administering a mixed formulation of a pharmaceutically effective
amount of an EGFR antagonist and a pharmaceutically effective
amount of an anti-c-Met antibody. In another particular embodiment,
the co-administration may conducted by separate simultaneous or
sequential administration of a pharmaceutically effective amount of
an EGFR antagonist and a pharmaceutically effective amount of an
anti-c-Met antibody. For sequential administration, the sequence of
administration (which active ingredient is administered first or
second) may be in any order. When the co-administration may
conducted by sequential administration, the interval between the
administration of an EGFR antagonist and the administration of an
anti-c-Met antibody may not be particularly limited. For example,
the EGFR antagonist and anti-C-met antibody can be sequentially
administered at a relatively short interval (e.g., the interval may
be 1 minute, 5 minutes, 10 minutes, 20 minutes, or 30 minutes), or
at a longer interval (e.g., the interval may be 1 hour, 5 hours, 12
hours, 1 day, 3 days, or 7 days).
[0033] According to the present invention, co-administration of a
drug targeting EGFR (EGFR antagonist) and an anti-c-Met antibody
may achieve excellent synergistic effects, compared to the case of
using a single drug.
[0034] In addition, the anti-c-Met antibody as defined below
exhibits c-Met degradation activity independently from Cbl which is
a typical RTK negative regulator. Thus, even if the anti-c-Met
antibody is administered to a patient in which EGFR antagonist
resistance occurs due to mutation in Cbl or insufficient Cbl
expression level, it may inhibit c-Met via another negative
regulator, LRIG1 which functions independently from Cbl. Therefore,
the co-administration of an anti-c-Met antibody and an EGFR
antagonist may be particularly useful for a patient where Cbl does
not function normally.
[0035] The "epidermal growth factor receptor (EGFR)" is a member of
HER family receptor tyrosine kinase (RTKs) consisting of EGFR
(HER1), HER2, HER3 and HER4. Binding of a ligand to an
extracellular domain of EGFR may induce receptor homo- or
hetero-dimerization with another ErbB receptor, causing
intracellular auto-phosphorylation of a tyrosine residue.
Auto-phosphorylation of EGFR leads a downstream signal transduction
network including activation of MAPK and PI3K/Akt affecting cell
proliferation, angiogenesis and metastasis. EGFR over-expression,
gene amplification, mutation, or rearrangement are frequently
observed in various kinds of human malignant cancers, and related
to poor prognosis of cancer treatment and poor clinical results.
For this reason, EGFR is an important target in anticancer therapy.
The EGFR may originate from a mammal, for instance, a primate such
as human or monkey, or a rodent such as mouse or rat, and the like.
For example, the EGFR may be a polypeptide encoded by nucleotide
sequence (mRNA) provided by GenBank Accession Nos. JQ739160,
JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166,
JQ739167, NM.sub.--005228.3, NM.sub.--201284.1, NM.sub.--201282.1,
or NM.sub.--201283.1, and the like.
[0036] The drug targeting EGFR (EGFR antagonist) may be at least
one selected from the group consisting of a small-molecule tyrosine
inhibitor such as erlotinib, gefitinib, and the like, and an
anti-EGFR antibody such as cetuximab, panitumumab, and the
like.
[0037] The term "c-Met" or "c-Met protein" refers to a receptor
tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF).
c-Met may be a c-Met protein from any species, particularly a
mammal, for instance, primates such as human c-Met (e.g.,
NP.sub.--000236) or monkey c-Met (e.g., Macaca mulatta,
NP.sub.--001162100), or rodents such as mouse c-Met (e.g.,
NP.sub.--032617.2) or rat c-Met (e.g., NP.sub.--113705.1), and the
like. The c-Met protein may include a polypeptide encoded by the
nucleotide sequence identified as GenBank Accession Number
NM.sub.--000245, a polypeptide having the amino acid sequence
identified as GenBank Accession Number NP.sub.--000236 or
extracellular domains thereof. The receptor tyrosine kinase c-Met
participates in various mechanisms, such as cancer development,
metastasis, migration of cancer cell, invasion of cancer cell,
angiogenesis, and the like.
[0038] Unless stated otherwise, the anti c-Met antibody included in
the pharmaceutical composition for combination therapy may refer to
not only a complete anti-c-Met antibody but also antigen-binding
fragments or variants of the antibody. The antigen-binding fragment
of the anti-c-Met antibody may refer to a fragment including an
antigen binding region of the anti-c-Met antibody, and can be
selected from the group consisting of a complementarity determining
region (CDR), fragment including CDR and Fc region, scFv,
(scFv).sub.2, Fab, Fab', and F(ab').sub.2 of the anti-c-Met
antibody. The variant of the antibody may be any isotype of
antibodies derived from human (e.g., IgG1, IgG2, IgG3, IgG4, and
the like) and other animals found in nature and/or one including
any Fc region of antibodies derived from human and other animals,
including a mutated hinge wherein at least one amino acid is
changed, deleted, inserted, or added.
[0039] The anti c-Met antibody may recognize a specific region of
c-Met, e.g., a specific region in the SEMA domain, as an epitope.
It may be any antibody or antigen-binding fragment that acts on
c-Met to induce c-Met intracellular internalization and
degradation.
[0040] c-Met, a receptor for hepatocyte growth factor (HGF), may be
divided into three portions: extracellular, transmembrane, and
intracellular. The extracellular portion is composed of an
.alpha.-subunit and a .beta.-subunit which are linked to each other
through a disulfide bond, and contains a SEMA domain responsible
for binding HGF, a PSI domain (plexin-semaphorins-integrin homology
domain) and an IPT domain (immunoglobulin-like fold shared by
plexins and transcriptional factors domain). The SEMA domain of
c-Met protein may have the amino acid sequence of SEQ ID NO: 79, an
extracellular domain that functions to bind HGF. A specific region
of the SEMA domain, that is, a region including the amino acid
sequence of SEQ ID NO: 71, which corresponds to amino acid residues
106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID
NO: 79) of c-Met protein, is a loop region between the second and
the third propellers within the epitopes of the SEMA domain. The
region acts as an epitope for the specific anti-c-Met antibody of
the present invention.
[0041] The term "epitope," as used herein, refers to an antigenic
determinant, a part of an antigen recognized by an antibody. In one
embodiment, the epitope may be a region including 5 or more
contiguous (consecutive or non-consecutive) amino acid residues
within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for
instance, 5 to 19 contiguous amino acid residues within the amino
acid sequence of SEQ ID NO: 71. For example, the epitope may be a
polypeptide including 5 to 19 contiguous amino acids selected from
among partial combinations of the amino acid sequence of SEQ ID NO:
71, wherein the polypeptide essentially includes the amino sequence
of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the
epitope. For example, the epitope may be a polypeptide including,
consisting essentially of, or consisting of the amino acid sequence
of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. The contiguous
amino acid residues of the polypeptide comprising the epitope may
refer to amino acid residues which are contiguous in the
polypeptide's primary, secondary, tertiary structure.
[0042] The epitope including the amino acid sequence of SEQ ID NO:
72 corresponds to the outermost part of the loop between the second
and third propellers within the SEMA domain of a c-Met protein. The
epitope including the amino acid sequence of SEQ ID NO: 73 is a
site to which the antibody or antigen-binding fragment according to
one embodiment most specifically binds.
[0043] Thus, the anti-c-Met antibody may specifically bind to an
epitope which has 5 to 19 contiguous amino acids within the amino
acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an
essential element. For example, the anti-c-Met antibody may
specifically bind to an epitope including the amino acid sequence
of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.
[0044] In one embodiment, the anti-c-Met antibody may be an
antibody or an antigen-binding fragment thereof, which includes:
(a) at least one heavy chain complementarity determining region
(CDR) selected from the group consisting of (a) a CDR-H1 including
the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the
amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid
sequence including 8-19 consecutive amino acids including amino
acid residues from the 3.sup.rd to 10.sup.th positions of SEQ ID
NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID
NO: 6, SEQ ID NO: 85, or an amino acid sequence including 6-13
consecutive amino acids including amino acid residues from the
1.sup.st to 6.sup.th positions of SEQ ID NO: 85, or a heavy chain
variable region including the at least one heavy chain
complementarity determining region; (b) at least one light chain
complementarity determining region (CDR) selected from the group
consisting of (a) a CDR-L1 including the amino acid sequence of SEQ
ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID
NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID
NO: 9, SEQ ID NO: 86, or an amino acid sequence including 9-17
consecutive amino acids including amino acid residues from the
1.sup.st to 9.sup.th positions of SEQ ID NO: 89, or a light chain
variable region including the at least one light chain
complementarity determining region; (c) a combination of the at
least one heavy chain complementarity determining region and the at
least one light chain complementarity determining region; or (e) a
combination of the heavy chain variable region and light chain
variable region.
[0045] Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are
respectively represented by following Formulas I to VI, below:
TABLE-US-00001 Formula I (SEQ ID NO: 4)
Xaa.sub.1-Xaa.sub.2-Tyr-Tyr-Met-Ser,
[0046] wherein Xaa.sub.1 is absent or Pro or Ser, and Xaa.sub.2 is
Glu or Asp,
TABLE-US-00002 Formula II (SEQ ID NO: 5)
Arg-Asn-Xaa.sub.3-Xaa.sub.4-Asn-Gly-Xaa.sub.5-Thr,
[0047] wherein Xaa.sub.3 is Asn or Lys, Xaa.sub.4 is Ala or Val,
and Xaa.sub.6 is Asn or Thr,
TABLE-US-00003 Formula III (SEQ ID NO: 6)
Asp-Asn-Trp-Leu-Xaa.sub.6-Tyr,
[0048] wherein Xaa.sub.6 is Ser or Thr,
TABLE-US-00004 Formula IV (SEQ ID NO: 7)
Lys-Ser-Ser-Xaa.sub.7-Ser-Leu-Leu-Ala-Xaa.sub.8-Gly-Asn-
Xaa.sub.9-Xaa.sub.10-Asn-Tyr-Leu-Ala
[0049] wherein Xaa.sub.7 is His, Arg, Gln, or Lys, Xaa.sub.8 is Ser
or Trp, Xaa.sub.9 is His or Gln, and Xaa.sub.10 is Lys or Asn,
TABLE-US-00005 Formula V (SEQ ID NO: 8)
Trp-Xaa.sub.11-Ser-Xaa.sub.12-Arg-Val-Xaa.sub.13
[0050] wherein Xaa.sub.11 is Ala or Gly, Xaa.sub.12 is Thr or Lys,
and Xaa.sub.13 is Ser or Pro, and
TABLE-US-00006 Formula VI (SEQ ID NO: 9)
Xaa.sub.14-Gln-Ser-Tyr-Ser-Xaa.sub.15-Pro-Xaa.sub.16-Thr
[0051] wherein Xaa.sub.14 is Gly, Ala, or Gln, Xaa.sub.15 is Arg,
His, Ser, Ala, Gly, or Lys, and Xaa.sub.16 is Leu, Tyr, Phe, or
Met.
[0052] In one embodiment, the CDR-H1 may have an amino acid
sequence selected from the group consisting of SEQ ID NOS: 1, 22,
23, and 24. The CDR-H2 may have an amino acid sequence selected
from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3
may have an amino acid sequence selected from the group consisting
of SEQ ID NOS: 3, 27, 28, and 85.
[0053] The CDR-L1 may have an amino acid sequence selected from the
group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106.
The CDR-L2 may have an amino acid sequence selected from the group
consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may have
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 12, 13, 14, 15, 16, 37, 86, and 89.
[0054] In another embodiment, the antibody or the antigen-binding
fragment may include a heavy variable region including a
polypeptide (CDR-H1) including an amino acid sequence selected from
the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a
polypeptide (CDR-H2) including an amino acid sequence selected from
the group consisting of SEQ ID NOS: 2, 25, and 26, and a
polypeptide (CDR-H3) including an amino acid sequence selected from
the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and a light
variable region including a polypeptide (CDR-L1) including an amino
acid sequence selected from the group consisting of SEQ ID NOS: 10,
29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an
amino acid sequence selected from the group consisting of SEQ ID
NOS 12, 13, 14, 15, 16, 37, 86, and 89.
[0055] Animal-derived antibodies produced by immunizing non-immune
animals with a desired antigen generally invoke immunogenicity when
injected to humans for the purpose of medical treatment, and thus
chimeric antibodies have been developed to inhibit such
immunogenicity. Chimeric antibodies are prepared by replacing
constant regions of animal-derived antibodies that cause an
anti-isotype response with constant regions of human antibodies by
genetic engineering. Chimeric antibodies are considerably improved
in an anti-isotype response compared to animal-derived antibodies,
but animal-derived amino acids still have variable regions, so that
chimeric antibodies have side effects with respect to a potential
anti-idiotype response. Humanized antibodies have been developed to
reduce such side effects. Humanized antibodies are produced by
grafting complementarity determining regions (CDR) which serve an
important role in antigen binding in variable regions of chimeric
antibodies into a human antibody framework.
[0056] The most important thing in CDR grafting to produce
humanized antibodies is choosing the optimized human antibodies for
accepting CDRs of animal-derived antibodies. Antibody databases,
analysis of a crystal structure, and technology for molecule
modeling are used. However, even when the CDRs of animal-derived
antibodies are grafted to the most optimized human antibody
framework, amino acids positioned in a framework of the
animal-derived CDRs affecting antigen binding are present.
Therefore, in many cases, antigen binding affinity is not
maintained, and thus application of additional antibody engineering
technology for recovering the antigen binding affinity is
necessary.
[0057] The anti c-Met antibodies may be mouse-derived antibodies,
mouse-human chimeric antibodies, humanized antibodies, or human
antibodies. The antibodies or antigen-binding fragments thereof may
be isolated from (i.e., not originally present in) a living body or
non-naturally occurring. The antibodies or antigen-binding
fragments thereof may be synthetic or monoclonal.
[0058] An intact antibody includes two full-length light chains and
two full-length heavy chains, in which each light chain is linked
to a heavy chain by disulfide bonds. The antibody has a heavy chain
constant region and a light chain constant region. The heavy chain
constant region is of a gamma (.gamma.), mu (.mu.), alpha
(.alpha.), delta (.delta.), or epsilon (.epsilon.) type, which may
be further categorized as gamma 1 (.gamma.1), gamma 2(.gamma.2),
gamma 3(.gamma.3), gamma 4(.gamma.4), alpha 1(.alpha.1), or alpha
2(.alpha.2). The light chain constant region is of either a kappa
(.kappa.) or lambda (.lamda.) type.
[0059] As used herein, the term "heavy chain" refers to full-length
heavy chain, and fragments thereof, including a variable region
V.sub.H that includes amino acid sequences sufficient to provide
specificity to antigens, and three constant regions, C.sub.H1,
C.sub.H2, and C.sub.H3, and a hinge. The term "light chain" refers
to a full-length light chain and fragments thereof, including a
variable region V.sub.L that includes amino acid sequences
sufficient to provide specificity to antigens, and a constant
region C.sub.L.
[0060] The term "complementarity determining region (CDR)" refers
to an amino acid sequence found in a hyper variable region of a
heavy chain or a light chain of immunoglobulin. The heavy and light
chains may respectively include three CDRs (CDRH1, CDRH2, and
CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact
residues that play an important role in the binding of antibodies
to antigens or epitopes. The terms "specifically binding" and
"specifically recognized" are well known to one of ordinary skill
in the art, and indicate that an antibody and an antigen
specifically interact with each other to lead to an immunological
activity.
[0061] The term "antigen-binding fragment" used herein refers to
fragments of an intact immunoglobulin including portions of a
polypeptide including antigen-binding regions having the ability to
specifically bind to the antigen. In one embodiment, the antibody
may be an antigen-binding fragment selected from the group
consisting of scFv, (scFv).sub.2, Fab, Fab', and F(ab').sub.2.
[0062] Among the antigen-binding fragments, Fab that includes light
chain and heavy chain variable regions, a light chain constant
region, and a first heavy chain constant region C.sub.H1, has one
antigen-binding site.
[0063] The Fab' fragment is different from the Fab fragment, in
that Fab' includes a hinge region with at least one cysteine
residue at the C-terminal of C.sub.H1.
[0064] The F(ab').sub.2 antibody is formed through disulfide
bridging of the cysteine residues in the hinge region of the Fab'
fragment. Fv is the smallest antibody fragment with only a heavy
chain variable region and a light chain variable region.
Recombination techniques of generating the Fv fragment are widely
known in the art.
[0065] Two-chain Fv includes a heavy chain variable region and a
light chain region which are linked by a non-covalent bond.
Single-chain Fv generally includes a heavy chain variable region
and a light chain variable region which are linked by a covalent
bond via a peptide linker or linked at the C-terminals to have a
dimer structure like the two-chain Fv.
[0066] The antigen-binding fragments may be attainable using
protease (for example, the Fab fragment may be obtained by
restricted cleavage of a whole antibody with papain, and the
F(ab').sub.2 fragment may be obtained by cleavage with pepsin), or
may be prepared by using a genetic recombination technique.
[0067] The term "hinge region," as used herein, refers to a region
between CH1 and CH2 domains within the heavy chain of an antibody
which functions to provide flexibility for the antigen-binding
site.
[0068] When an animal antibody undergoes a chimerization process,
the IgG1 hinge of animal origin may be replaced with a human IgG1
hinge or IgG2 hinge while the disulfide bridges between two heavy
chains are reduced from three to two in number. In addition, an
animal-derived IgG1 hinge is shorter than a human IgG1 hinge.
Accordingly, the rigidity of the hinge is changed. Thus, a
modification of the hinge region may bring about an improvement in
the antigen binding efficiency of the humanized antibody. The
modification of the hinge region through amino acid deletion,
addition, or substitution is well-known to those skilled in the
art.
[0069] In one embodiment, the anti-c-Met antibody or an
antigen-binding fragment thereof may be modified by the deletion,
insertion, addition, or substitution of at least one amino acid
residue on the amino acid sequence of the hinge region so that it
exhibit enhanced antigen-binding efficiency. For example, the
antibody may include a hinge region including the amino acid
sequence of SEQ ID NO: 100(U7-HC6), 101(U6-HC7), 102(U3-HC9),
103(U6-HC8), or 104(U8-HC5), or a hinge region including the amino
acid sequence of SEQ ID NO: 105 (non-modified human hinge).
Preferably, the hinge region has the amino acid sequence of SEQ ID
NO: 100 or 101.
[0070] In one embodiment of the anti-c-Met antibody or
antigen-binding fragment, the variable domain of the heavy chain
has the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92,
93, or 94 and the variable domain of the light chain has the amino
acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98,
99, or 107.
[0071] In one embodiment, the anti-c-Met antibody may be a
monoclonal antibody. The monoclonal antibody may be produced by the
hybridoma cell line deposited with Accession No. KCLRF-BP-00220,
which binds specifically to the extracellular region of c-Met
protein (refer to Korean Patent Publication No. 2011-0047698, the
entire disclosure of which is incorporated herein by reference).
The anti-c-Met antibody may include all the antibodies defined in
Korean Patent Publication No. 2011-0047698.
[0072] By way of further example, the anti-c-Met antibody or the
antibody fragment may include:
[0073] a heavy chain including the amino acid sequence selected
from the group consisting of the amino acid sequence of SEQ ID NO:
62 (wherein the amino acid sequence from amino acid residues from
the 1.sup.st to 17.sup.th positions is a signal peptide), or the
amino acid sequence from the 18.sup.th to 462.sup.nd positions of
SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein
the amino acid sequence from the 1.sup.st to 17.sup.th positions is
a signal peptide), the amino acid sequence from the 18.sup.th to
461.sup.st positions of SEQ ID NO: 64, the amino acid sequence of
SEQ ID NO: (wherein the amino acid sequence from the 1.sup.st to
17.sup.th positions is a signal peptide), and the amino acid
sequence from the 18.sup.th to 460.sup.th positions of SEQ ID NO:
66; and
[0074] a light chain including the amino acid sequence selected
from the group consisting of the amino acid sequence of SEQ ID NO:
68 (wherein the amino acid sequence from the 1.sup.st to 20.sup.th
positions is a signal peptide), the amino acid sequence from the
21.sup.st to 240.sup.th positions of SEQ ID NO: 68, the amino acid
sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the
1.sup.st to 20.sup.th positions is a signal peptide), the amino
acid sequence from the 21.sup.st to 240.sup.th positions of SEQ ID
NO: 70, and the amino acid sequence of SEQ ID NO: 108.
[0075] For example, the anti-c-Met antibody may be selected from
the group consisting of:
[0076] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 62 or the amino acid sequence from the
18.sup.th to 462.sup.nd positions of SEQ ID NO: 62 and a light
chain including the amino acid sequence of SEQ ID NO: 68 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 68;
[0077] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 64 or the amino acid sequence from the
18.sup.th to 461.sup.st positions of SEQ ID NO: 64 and a light
chain including the amino acid sequence of SEQ ID NO: 68 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 68;
[0078] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 66 or the amino acid sequence from the
18.sup.th to 460.sup.th positions of SEQ ID NO: 66 and a light
chain including the amino acid sequence of SEQ ID NO: 68 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 68;
[0079] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 62 or the amino acid sequence from the
18.sup.th to 462.sup.nd positions of SEQ ID NO: 62 and a light
chain including the amino acid sequence of SEQ ID NO: 70 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 70;
[0080] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 64 or the amino acid sequence from the
18.sup.th to 461.sup.st positions of SEQ ID NO: 64 and a light
chain including the amino acid sequence of SEQ ID NO: 70 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 70;
[0081] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 66 or the amino acid sequence from the
18.sup.th to 460.sup.th positions of SEQ ID NO: 66 and a light
chain including the amino acid sequence of SEQ ID NO: 70 or the
amino acid sequence from the 21.sup.st to 240.sup.th positions of
SEQ ID NO: 70;
[0082] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 62 or the amino acid sequence from the
18.sup.th to 462.sup.nd positions of SEQ ID NO: 62 and a light
chain including the amino acid sequence of SEQ ID NO: 108;
[0083] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 64 or the amino acid sequence from the
18.sup.th to 461.sup.st positions of SEQ ID NO: 64 and a light
chain including the amino acid sequence of SEQ ID NO: 108; and
[0084] an antibody including a heavy chain including the amino acid
sequence of SEQ ID NO: 66 or the amino acid sequence from the
18.sup.th to 460.sup.th positions of SEQ ID NO: 66 and a light
chain including the amino acid sequence of SEQ ID NO: 108.
[0085] The polypeptide of SEQ ID NO: 70 is a light chain including
human kappa (K) constant region, and the polypeptide with the amino
acid sequence of SEQ ID NO: 68 is a polypeptide obtained by
replacing histidine at position 62 (corresponding to position 36 of
SEQ ID NO: 68 according to kabat numbering) of the polypeptide with
the amino acid sequence of SEQ ID NO: 70 with tyrosine. The
production yield of the antibodies may be increased by the
replacement. The polypeptide with the amino acid sequence of SEQ ID
NO: 108 is a polypeptide obtained by replacing serine at position
32 (position 27e according to kabat numbering in the amino acid
sequence from amino acid residues 21 to 240 of SEQ ID NO: 68;
positioned within CDR-L1) of SEQ ID NO: 108 with tryptophan. By
such replacement, antibodies and antibody fragments including such
sequences exhibits increased activities, such as c-Met biding
affinity, c-Met degradation activity, Akt phosphorylation
inhibition, and the like.
[0086] In another embodiment, the anti c-Met antibody may include a
light chain complementarity determining region including the amino
acid sequence of SEQ ID NO: 106, a variable domain of a light chain
including the amino acid sequence of SEQ ID NO: 107, or a light
chain including the amino acid sequence of SEQ ID NO: 108.
[0087] According to one particular embodiment, the antibody may act
as an antagonist of c-Met protein.
[0088] As used herein, the term "antagonist" is intended to
encompass all molecules that at least partially block, suppress, or
neutralize at least one of the biological activities of a target
(e.g., c-Met). By way of example, an "antagonist" antibody means an
antibody that represents suppression or inhibition against the
biological activity of the antigen to which the antibody binds
(e.g., c-Met). An antagonist may function to reduce ligand-induced
receptor phosphorylation or to incapacitate or kill cells which
have been activated by ligands. Also, an antagonist may interfere
with receptor-ligand interaction or substantially reduce the
interaction by changing the three-dimensional structure of the
receptor or by down regulation.
[0089] According to another particular embodiment, the anti-c-Met
antibody may be a bispecific antibody wherein an anti-c-Met
antibody or an antigen-binding fragment thereof is coupled with an
antigen-binding fragment binding to an antigen other than
c-Met.
[0090] For example, the antigen other than c-Met may be vascular
endothelial cell growth factor (VEGF), wherein the VEGF may be a
polypeptide encoded by nucleotide sequences (mRNA) provided by
GenBank Accession Number NM.sub.--001025366.2,
NM.sub.--001025367.2, NM.sub.--001025368.2, NM.sub.--001025369.2,
NM.sub.--001025370.2, NM.sub.--001033756.2, NM.sub.--001171622.1,
NM.sub.--001171623.1, NM.sub.--001171624.1, NM.sub.--001171625.1,
NM.sub.--001171626.1, NM.sub.--001171627.1, NM.sub.--001171628.1,
NM.sub.--001171629.1, NM.sub.--001171630.1, NM.sub.--001204384.1,
NM.sub.--001204385.1, NM.sub.--003376.5, and the like. The
antigen-binding fragment binding to antigens other than c-Met may
be a VEGF-binding fragment, which may be a VEGF-binding molecule,
for example, a VEGF receptor, VEGF-binding protein such as
anti-VEGF antibody, and the like, or a fragment including the
VEGF-binding region of the VEGF-binding protein, for example, an
antigen-binding fragment of anti-VEGF antibody or a VEGF-binding
portion of a VEGF receptor. For example, the anti-VEGF antibody may
be Bevacizumab, and the antigen-binding fragment of the anti-VEGF
antibody may be selected from the group consisting of a
complementarity determining regions (CDRs), a combination of CDR
and Fc regions, scFv, (scFv).sub.2, Fab, Fab' or F(ab').sub.2 and
the like of the anti-VEGF antibody. The VEGF receptor may be, for
example, human VEGF Receptor 1 (P17948.2; SEQ ID NO: 113), human
VEGF Receptor 2 (P35968.2), human VEGF Receptor 3 (P35916.3), and
the like. And, the fragment including a VEGF-binding portion of the
VEGF-binding protein may be a second Ig-like domain 2 (VIG2) or a
polypeptide including a VIG2 portion of a VEGF receptor. For
example, a fragment including a VEGF-binding portion of human VEGF
receptor 1 may be a second Ig-like domain 2 (VIG2; for example,
129th to 229th amino acid sequence (SEQ ID NO: 114) of P17948.2
amino acid sequence (SEQ ID NO: 113)), or a polypeptide including
continuous 102 to 1338 amino acids including the VIG2 in the SEQ ID
NO: 113.
[0091] Thus, according to one particular embodiment, the
VEGF-binding fragment may be an anti-VEGF antibody such as
Bevacizumab, an antigen-binding fragment thereof selected from the
group consisting of a complementary determining region (CDR), a
combination of CDR and Fc region, scFv, (scFv).sub.2, Fab, Fab' or
F(ab').sub.2, and the like, a VEGF receptor (for example, SEQ ID
NO: 113), or a VEGF-binding portion of the VEGF receptor (for
example, VIG2 of SEQ ID NO: 114, a polypeptide including continuous
102 to 1338 amino acids including VIG2 of SEQ ID NO: 114 in SEQ ID
NO: 113), and the like.
[0092] According to a particular embodiment, the bispecific
antibody may be those wherein the VEG binding fragment, for
example, VIG2 is coupled to the Fc region of the anti-c-Met
antibody, an antigen-binding fragment thereof, or a modified form
(variant) thereof. The anti-c-Met antibody, an antigen-binding
fragment thereof, or a variant thereof may be coupled to the
VEGF-binding fragment through a linker. The linker may be a peptide
linker including 1 to 100, specifically 2 to 50, more specifically
5 to 30 amino acid lengths. For example, the peptide linker may
include at least one amino acid selected from the group consisting
of Gly, Asn, Ser, Thr, Ala, and the like, but not be limited
thereto. In one particular embodiment, the linker may be
represented by (GGGGS).sub.n, wherein n is a repeat number of
(GGGGS) unit, and it may be 1 to about 10, specifically 1 to about
5, considering efficiency of the bispecific (dual-targeting)
antibody (see FIG. 7).
[0093] In particular, the pharmaceutical composition may be
formulated into an immunoliposome since it contains an antibody or
an antigen-binding fragment. A liposome containing an antibody may
be prepared using any methods well known in the art. The
immunnoliposome may be a lipid composition including
phosphatidylcholine, cholesterol, and polyethyleneglycol-derived
phosphatidylethanolamine, and may be prepared by a reverse phase
evaporation method. For example, Fab' fragments of an antibody may
be conjugated to the liposome through a disulfide-exchange
reaction. A chemical drug, such as doxorubicin, may be further
included in the liposome.
[0094] The combined mixture obtained by mixing a pharmaceutically
effective amount of an EGFR antagonist and a pharmaceutically
effective amount of an anti-c-Met antibody, a first pharmaceutical
composition including a pharmaceutically effective amount of an
EGFR antagonist as an active ingredient, or a second pharmaceutical
composition including a pharmaceutically effective amount of an
anti-c-Met antibody as an active ingredient may be provided
together with a pharmaceutically acceptable carrier, diluent,
and/or excipient.
[0095] The pharmaceutically acceptable carriers that may be
included in the combined mixture or the pharmaceutical compositions
may be those commonly used in formulations of drugs, and may be,
but not limited to, at least one selected from the group consisting
of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum
acacia, calcium phosphate, alginates, gelatin, calcium silicate,
micro-crystalline cellulose, polyvinylpyrrolidone, cellulose,
water, syrup, methyl cellulose, methylhydroxy benzoate,
propylhydroxy benzoate, talc, magnesium stearate, and mineral oil.
Besides these components, the combined mixture or the
pharmaceutical compositions may further include at least one
selected from the group consisting of a diluent, an excipient, a
lubricant, a wetting agent, a sweetener, a flavor enhancer, an
emulsifying agent, a suspension agent, and a preservative.
[0096] The mixture or the pharmaceutical compositions may be
administered orally or parenterally. Parenteral administration may
include intravenous injection, subcutaneous injection, muscular
injection, intraperitoneal injection, endothelial administration,
local administration, intranasal administration, intrapulmonary
administration, and rectal administration. Since oral
administration leads to digestion of proteins or peptides, an
active ingredient in the compositions for oral administration must
be coated or formulated to prevent digestion in stomach. In
addition, the compositions may be administered using an optional
device that enables an active substance to be delivered to target
cells.
[0097] The term "the pharmaceutically effective amount" as used in
this specification refers to an amount at which each active
ingredient can exert pharmaceutically significant effects.
[0098] For single dose (for one-time administration), a
pharmaceutically effective amount of the EGFR antagonist and a
pharmaceutically effective amount of the anti-c-Met antibodies may
be prescribed in various ways, depending on many factors including
formulation methods, administration manners, ages, body weight,
gender, pathologic conditions, diets of patients, administration
time, administration interval, administration route, excretion
speed, and reaction sensitivity. For example, the effective amount
of the EGFR antagonist for single dose may be, but not limited to,
in the range of 0.01 to 100 mg/kg, particularly 0.2 to 10 mg/kg,
and the effective amount of the anti-c-Met antibody for single dose
may be in the range of 0.01 to 100 mg/kg, particularly 0.2 to 10
mg/kg. The effective amount for single dose may be formulated into
a single formulation in a unit dosage form or formulated in
suitably divided dosage forms, or it may be manufactured to be
contained in a multiple dosage container. For the kit, the
effective amount of the EGFR antagonist and the effective amount of
the anti-c-Met antibody for single dose may be contained in a
package container as a base unit.
[0099] The co-administration interval between the administrations
is defined as a period between the first administration and the
following administration. The administration interval may be any
length of time including, but not limited to, 5 hours to 30 days
(e.g., 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days,
5 days, 6, days, 7 days, 10 days, 14 days, 21 days, or 28 days),
and particularly 7 days to 14 days. For the combined therapy, the
first pharmaceutical composition containing a pharmaceutically
effective amount of an EGFR antagonist as an active ingredient, and
the second pharmaceutical composition containing a pharmaceutically
effective amount of an anti-c-Met antibody or an antigen-binding
fragment thereof as an active ingredient may be co-administered in
a given time interval (e.g., several minutes, several hours or
several days, or several weeks) to be determined by a type of
diseases, a patient's conditions, etc. For example, the first
pharmaceutical composition and the second pharmaceutical
composition may be simultaneously administered (administration
interval within 1 minute) or sequentially administered
(administration interval of 1 minute or over), and in case of
sequential administration, the administration interval between the
first pharmaceutical composition and the second pharmaceutical
composition may be 1 minute to 30 days, particularly, 1 minute to 7
days, 1 minute to 24 hours, or 1 minute to 60 minutes, and more
particularly, 1 minute to 10 minutes, and their administration
order may be reversed.
[0100] The combined mixture or the pharmaceutical compositions may
be a solution in oil or an aqueous medium, a suspension, a syrup,
an emulsifying solution form, or they may be formulated into a form
of an extract, elixirs, powders, granules, a tablet or a capsule,
and they may further include a dispersing agent or a stabilizing
agent for their formulation. The combined mixture or the
pharmaceutical composition may be formulated as a unit dosage form
using a pharmaceutically acceptable carrier and/or excipient, or it
may be formulated to be contained into a multiple dosage container,
according to a method that can be easily practiced by one of
ordinary knowledge in the art.
[0101] The patients may be mammals including primates such as
humans and monkeys and rodents such as mice and rats. Furthermore,
the patients may be cancer patients, or patients having EGFR
antagonist resistance. In a particular embodiment, if c-Met gene
copy number and/or expression amount increases 2 times or more, or
3 times or more, specifically 2 to 5 times or 3 to 5 times in a
cell sample, compared to normal cells, it may be judged that EGFR
antagonist resistance occurs. The normal cells are those wherein
c-Met-related diseases including tumors do not occur, and may be
those originated from the same tissue as the cell sample to be
tested, wherein c-Met-related diseases including tumors do not
occur.
[0102] In one embodiment, the patients may be those where existing
c-Met antibody does not exhibit c-Met degradation activities
because Cbl is not present or it is present at a low concentration
(for example, when Cbl is subject to immunohistochemistry staining
using an anti-Cbl antibody available for immunohistochemistry
staining, it is present at a concentration corresponding to `+1` or
`-`), a functional mutation of Cbl is induced, or an interaction
site of c-Met with Cbl is mutated. In addition, the patients may be
those capable of degrading c-Met by their intrinsic c-Met antibody
via an independent pathway from Cbl by a mediation of LRIG1 due to
a high expression amount of LRIG1.
[0103] Therefore, the prevention and/or treatment method may
further include a step of identifying a patient with inactivated
Cbl and/or high expression amount of LRIG1, before the
co-administration step.
[0104] The step of identifying the patients may include:
[0105] (1) a step of identifying a Cbl concentration in a cell
specimen isolated from patients, whether Cbl is mutated or not,
and/or whether an interaction site of c-Met with Cbl is mutated or
not; and
[0106] (2) a step of determining, in cases that the Cbl
concentration falls under `+1` or `-` when it is subject to
immunohistochemistry staining using an anti Cbl antibody available
for immunohistochemistry staining, a Cbl mutation is present,
and/or a mutation at the interaction site of c-Met with Cbl is
present, that these cells or a patient from which the cells are
derived, are suitable subjects for administration of the
pharmaceutical compositions for combined therapy.
[0107] In a particular embodiment, the step of identifying a
patient may further include a step of identifying an LRIG1
concentration in a cell specimen isolated from patients, and when
the LRIG1 concentration falls under +2 or +3 when it is subject to
immunohistochemistry staining using an anti-LRIGI antibody
available for immunohistochemistry staining, a step of determining
that the patient is suitable for administration of the
pharmaceutical compositions for combined therapy.
[0108] "Cbl," "Cbl proteins," or "Cbl enzymes" are also referred to
E3 ligase, a protein involved in a cell signal transduction and
protein ubiquitination. The proteins function in the degradation of
c-Met proteins by internalizing them within cells. The proteins may
be polypeptides encoded by nucleotide sequences deposited under
GenBank Accession Numbers NM.sub.--005188, NM.sub.--007619,
NM.sub.--170662, or NM.sub.--001033238, or polypeptides having
amino acid sequences of GenBank Accession Numbers NP.sub.--005199,
NP.sub.--031645, NP.sub.--733762, or NP.sub.--001028410.
[0109] "LRIG1" (leucine-rich repeats and immunoglobulin-like
domains protein 1) is a transmembrane protein that interacts with
receptor tyrosine kinases such as EGFR-class, MET, and RET
proteins. LRIG1 may be derived from mammals including primates such
as humans and monkeys and rodents such as mice and rats and in
particular, it may be human LRIG1 (Accession No. NM.sub.--015541 or
NP.sub.--056356).
[0110] The identification of Cbl concentration or LRIG1
concentration may be carried out by measuring the concentration by
any known protein quantity analysis means, and/or evaluating the
measured results. For example, Cbl concentration or LRIG1
concentration may be measured through any known enzyme reaction,
fluorescence, luminescence, and/or radiation detection using an
antibody or an aptamer specifically binding to Cbl or LRIG1,
respectively. In particular, the concentration may be measured by a
method selected from the group consisting of immunochromatography,
immunohistochemistry staining, enzyme linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA),
fluorescence immunoassay (FIA), luminescence immunoassay (LIA), and
western blotting, but it is not limited thereto. The detection
substances for the measurement of Cbl concentration or LRIG1
concentration may be at least one selected from the group
consisting of an antibody, an aptamer, etc. specifically binding to
Cbl or LRIG1.
[0111] The Cbl mutations may be any mutations at Cbl genes that
cause a loss in functions associated with an interaction with c-Met
(e.g., binding), and/or the cell internalization of c-Met and/or
the degradation of c-Met, and/or any sequential or structural
mutations of Cbl proteins. In a particular embodiment, the Cbl
mutations may be a deletion of a successive 51 or more nucleotides
(for example, 51 to 200 nucleotides) or a substitution with
different nucleotides within a region from residues 1169 to 1414
from nucleotide sequences deposited under GenBank Accession Number
NM.sub.--005188. Alternatively, the Cbl mutations may be a deletion
of 17 or more consecutive amino acids (for example, 17 to 100
consecutive amino acids) or a substitution with different amino
acids within a region from residues 343 to 424 from the amino acid
sequences of GenBank Accession Number NP.sub.--005179. Such
mutations induce the modification of RING Finger Motif of Cbl and
result in function loss as an E3 ligase enzyme. Thus, the ability
to degrade other proteins vanishes due to the mutations of these
nucleotides or amino acids.
[0112] Whether such Cbl mutations occur or not may be identified by
direct analysis of nucleotide sequences or amino acid sequences, by
measuring them via RT-PCR or DNA sequencing methods, etc., and/or
by evaluating the measured results, but not limited thereto.
[0113] A substance capable of detecting Cbl mutations may be at
least one selected from the group consisting of a primer capable of
detecting such mutations, an anti-Cbl antibody or an aptamer
specifically binding to Cbl, etc., but not limited thereto. The
primers capable of detecting Cbl mutations may be successive 20 to
50 sequences containing mutated sites among the nucleotide
sequences of mutated Cbl genes and/or sequences complementary
thereto or sequences having 80% or more, particularly 90% or more,
and more particularly 95% or more of sequence identity/homology
that can hybridize therewith.
[0114] The c-Met mutations refer to mutations of c-Met occurring at
a site recognized and/or bound by Cbl, and encompass mutations that
prevent Cbl from interacting with c-Met (e.g., binding) although
Cbl is sufficiently present in quantities or no function loss
change occurs.
[0115] "The interaction site of c-Met with Cbl" is a site
recognized and interacted by Cbl among the structures of c-Met and
it enables the intracellular migration and degradation of c-Met by
Cbl. The typical interaction site of c-Met with Cbl may be the
1003.sup.th amino acid residue, tyrosine (Y1003), which is an
interaction site with Cbl, or a site encoded by exon 14 of c-Met
genes. The exon 14 region of c-Met genes may be a site from
residues 3075 to 3215 from the full-length nucleotide sequences of
GenBank Accession No. NM.sub.--000245, or a site from residues 964
to 1009 from the full-length amino acid sequences of GenBank
Accession No. NP.sub.--000236. The c-Met mutations may be a
deletion of the 1003.sup.th amino acid residue, tyrosine (Y1003),
from c-Met, or a substitution with other amino acids (for example,
amino acid selected from the group consisting of alanine,
isoleucine, leucine, methionine, phenylalanine, proline,
tryptophan, valine, asparagine, cysteine, glutamine, glycine,
serine, threonine, aspartate, glutamate, arginine, histidine and
lysine, and particularly, phenylalanine), or a deletion of a 141 or
more consecutive nucleotides (for example, a 141 to 300 consecutive
nucleotides) from exon 14 region of the c-Met genes, or a
substitution with other nucleotides. Additionally or alternatively,
the c-Met mutations may be a deletion of 46 or more consecutive
amino acids (for example, a 46 to 100 consecutive amino acids) from
the polypeptide encoded by exon 14 region or a substitution with
other amino acids. In a particular embodiment, the c-Met mutations
may be a deletion of the 1003.sup.th amino acid residue, tyrosine
(Y1003), of c-Met or a substitution with phenylalanine (that is,
Y1003F), a deletion of the exon 14 region of the c-Met genes, or a
deletion of polypeptides encoded by the exon 14 region from the
c-Met proteins.
[0116] Whether such c-Met mutations occur or not may be identified
by direct analysis of nucleotide sequences or amino acid sequences,
by measuring them via RT-PCR or DNA sequencing methods, etc.,
and/or by evaluating the measured results, but not limited thereto.
A substance capable of detecting c-Met mutations may be one or more
selected from the group consisting of a primer, a probe and an
aptamer, which is capable of detecting such mutations (sequences of
20 to 50 amino acids containing mutated sites among the nucleotide
sequences of mutated Cbl genes and/or sequences complementary
thereto or sequences having 80% or more, particularly 90% or more,
and more particularly 95% or more of sequence identity/homology
that can hybridize therewith), an antibody or an aptamer
specifically binding to mutated c-Met, etc., but not limited
thereto.
[0117] The pharmaceutical compositions for combination therapy
according to the present invention may be used for prevention
and/or treatment of c-Met and EGFR induced diseases, for example,
disease induced by increase in c-Met copy number and/or expression
amount, typically cancers. The cancers may be, although not limited
thereto, at least one selected from the group consisting of
squamous cell carcinoma, small-cell lung cancer, non-small-cell
lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of
the lung, peritoneal carcinoma, skin cancer, melanoma in the skin
or eyeball, rectal cancer, cancer near the anus, esophagus cancer,
small intestinal tumor, endocrine gland cancer, parathyroid cancer,
adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or
acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatocellular adenoma,
breast cancer, colon cancer, large intestine cancer, endometrial
carcinoma or uterine carcinoma, salivary gland tumor, kidney
cancer, prostate cancer, vulvar cancer, thyroid cancer, head and
neck cancers, and the like.
[0118] The prevention and/or treatment effects of the cancers may
include effects of not only suppressing the growth of the cancer
cells but also suppressing the progression of cancers due to
migration, invasion, metastasis thereof, and the like.
[0119] According to another particular embodiment, the c-Met and
EGFR induced disease for which the composition for combination
therapy of the present invention may be applied may be kidney
diseases such as autosomal dominant polycystic kidney disease
(ADPKD).
[0120] The combination therapy of an EGFR antagonist and an
anti-c-Met antibody according to the present invention enables
effective anticancer treatment even for cancer cells where EGFR
antagonist resistance occurs. By the combination therapy, excellent
anticancer activity may be expected in cancer cells where
anticancer effects may not be achieved or may be insignificant by
administration of an EGFR antagonist or an anti-c-Met antibody
alone.
[0121] The pharmaceutical composition for combination therapy
including an EGFR antagonist and an anti-c-Met antibody may achieve
the following effects:
[0122] 1. Excellent anticancer effects on cancer cells where
anticancer effects may not be achieved or may be insignificant by
single administrations of an EGFR antagonist and an anti-c-Met
antibody,
[0123] 2. Anticancer effects on cancers where EGFR antagonist
resistance occurs and thus effects may not be achieved by the
administration of EGFR antagonist alone,
[0124] 3. Anticancer effects through effective inhibition of EGFR
and c-Met via a route mediated by LRIG1 independently from Cbl in
cancers having high c-Met expression amount due to EGFR antagonist
resistance,
[0125] 4. Prevention and/or treatment effect on other diseases in
which c-Met/HGF signal transduction system and EGF/EGFR signal
transduction system are involved, besides cancers.
[0126] Hereafter, the present invention will be described in detail
by examples. The following examples are intended merely to
illustrate the invention and are not construed to restrict the
invention.
EXAMPLES
Reference Example 1
Construction of Anti-c-Met Antibody
[0127] 1.1. Production of "AbF46", a Mouse Antibody to c-Met
[0128] 1.1.1. Immunization of Mouse
[0129] To obtain immunized mice necessary for the development of a
hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4
to 6 weeks old, was intraperitoneally injected with a mixture of
100 .mu.g of human c-Met/Fc fusion protein (R&D Systems) and
one volume of complete Freund's adjuvant. Two weeks after the
injection, a second intraperitoneal injection was conducted on the
same mice with a mixture of 50 .mu.g of human c-Met/Fc protein and
one volume of incomplete Freund's adjuvant. One week after the
second immunization, the immune response was finally boosted. Three
days later, blood was taken from the tails of the mice and the sera
were 1/1000 diluted in PBS and used to examine a titer of antibody
to c-Met by ELISA. Mice found to have a sufficient antibody titer
were selected for use in the cell fusion process.
[0130] 1.1.2. Cell Fusion and Production of Hybridoma
[0131] Three days before cell fusion, BALB/c mice (Japan SLC, Inc.)
were immunized with an intraperitoneal injection of a mixture of 50
.mu.g of human c-Met/Fc fusion protein and one volume of PBS. The
immunized mice were anesthetized before excising the spleen from
the left half of the body. The spleen was meshed to separate
splenocytes which were then suspended in a culture medium (DMEM,
GIBCO, Invitrogen). The cell suspension was centrifuged to recover
the cell layer. The splenocytes thus obtained (1.times.10.sup.8
cells) were mixed with myeloma cells (Sp2/0) (1.times.10.sup.8
cells), followed by spinning to give a cell pellet. The cell pellet
was slowly suspended, treated with 45% polyethylene glycol (PEG) (1
mL) in DMEM for 1 min at 37.degree. C., and supplemented with 1 mL
of DMEM. To the cells was added 10 mL of DMEM over 10 min, after
which incubation was conducted in a water bath at 37.degree. C. for
5 min. Then the cell volume was adjusted to 50 mL before
centrifugation. The cell pellet thus formed was resuspended at a
density of 1.about.2.times.10.sup.5 cells/mL in a selection medium
(HAT medium) and 0.1 mL of the cell suspension was allocated to
each well of 96-well plates which were then incubated at 37.degree.
C. in a CO.sub.2 incubator to establish a hybridoma cell
population.
[0132] 1.1.3. Selection of Hybridoma Cells Producing Monoclonal
Antibodies to c-Met Protein
[0133] From the hybridoma cell population established in Reference
Example 1.1.2, hybridoma cells which showed a specific response to
c-Met protein were screened by ELISA using human c-Met/Fc fusion
protein and human Fc protein as antigens.
[0134] Human c-Met/Fc fusion protein was seeded in an amount of 50
.mu.L (2 .mu.g/mL)/well to microtiter plates and allowed to adhere
to the surface of each well. The antibody that remained unbound was
removed by washing. For use in selecting the antibodies that do not
bind c-Met but recognize Fc, human Fc protein was attached to the
plate surface in the same manner.
[0135] The hybridoma cell culture obtained in Reference Example
1.1.2 was added in an amount of 50 .mu.L to each well of the plates
and incubated for 1 hour. The cells remaining unreacted were washed
out with a sufficient amount of Tris-buffered saline and Tween 20
(TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added
to the plates and incubated for 1 hour at room temperature. The
plates were washed with a sufficient amount of TBST, followed by
reacting the peroxidase with a substrate (OPD). Absorbance at 450
nm was measured on an ELISA reader.
[0136] Hybridoma cell lines which secrete antibodies that
specifically and strongly bind to human c-Met but not human Fc were
selected repeatedly. From the hybridoma cell lines obtained by
repeated selection, a single clone producing a monoclonal antibody
was finally separated by limiting dilution. The single clone of the
hybridoma cell line producing the monoclonal antibody was deposited
with the Korean Cell Line Research Foundation, an international
depository authority located at Yungun-Dong, Jongno-Gu, Seoul,
Korea, on Oct. 9, 2009, with Accession No. KCLRF-BP-00220 according
to the Budapest Treaty (refer to Korean Patent Laid-Open
Publication No. 2011-0047698).
[0137] 1.1.4. Production and Purification of Monoclonal
Antibody
[0138] The hybridoma cell line obtained in Reference Example 1.1.3
was cultured in a serum-free medium, and the monoclonal antibody
(AbF46) was produced and purified from the cell culture.
[0139] First, the hybridoma cells cultured in 50 mL of a medium
(DMEM) supplemented with 10% (v/v) FBS were centrifuged and the
cell pellet was washed twice or more with 20 mL of PBS to remove
the FBS therefrom. Then, the cells were resuspended in 50 mL of
DMEM and incubated for 3 days at 37.degree. C. in a CO.sub.2
incubator.
[0140] After the cells were removed by centrifugation, the
supernatant was stored at 4.degree. C. before use or immediately
used for the separation and purification of the antibody. An AKTA
system (GE Healthcare) equipped with an affinity column (Protein G
agarose column; Pharmacia, USA) was used to purify the antibody
from 50 to 300 mL of the supernatant, followed by concentration
with a filter (Amicon). The antibody in PBS was stored before use
in the following examples.
[0141] 1.2. Construction of chAbF46, a Chimeric Antibody to
c-Met
[0142] A mouse antibody is apt to elicit immunogenicity in humans.
To solve this problem, chAbF46, a chimeric antibody, was
constructed from the mouse antibody AbF46 produced in Experimental
Example 1.1.4 by replacing the constant region, but not the
variable region responsible for antibody specificity, with an amino
sequence of the human IgG1 antibody.
[0143] In this regard, a gene was designed to include the
nucleotide sequence of "EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI"
(SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of
"EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI" (SEQ ID NO: 39) for a
light chain and synthesized. Then, a DNA fragment having the heavy
chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having
the light chain nucleotide sequence (SEQ ID NO: 39) were digested
with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into
a pOptiVEC.TM.-TOPO TA Cloning Kit enclosed in an OptiCHO.TM.
Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a
pcDNA.TM. 3.3-TOPO TA Cloning Kit (Cat no. 8300-01),
respectively.
[0144] Each of the constructed vectors was amplified using Qiagen
Maxiprep kit (Cat no. 12662), and a transient expression was
performed using Freestyle.TM. MAX 293 Expression System
(Invitrogen). 293 F cells were used for the expression and cultured
in FreeStyle.TM. 293 Expression Medium in a suspension culture
manner. At one day before the transient expression, the cells were
provided in the concentration of 5.times.10.sup.5 cells/ml, and
after 24 hours, when the cell number reached to 1.times.10.sup.6
cells/ml, the transient expression was performed. A transfection
was performed by a liposomal reagent method using Freestyle.TM. MAX
reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided
in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and
mixed with 2 ml of OptiPro.TM. SFM (invtrogen) (A), and in another
15 ml tube, 100 ul (microliter) of Freestyle.TM. MAX reagent and 2
ml of OptiPro.TM. SFM were mixed (B), followed by mixing (A) and
(B) and incubating for 15 minutes. The obtained mixture was slowly
mixed with the cells provided one day before the transient
expression. After completing the transfection, the cells were
incubated in 130 rpm incubator for 5 days under the conditions of
37.degree. C., 80% humidity, and 8% CO.sub.2.
[0145] Afterwards, the cells were incubated in DMEM supplemented
with 10% (v/v) FBS for 5 hours at 37.degree. C. under a 5% CO.sub.2
condition and then in FBS-free DMEM for 48 hours at 37.degree. C.
under a 5% CO.sub.2 condition.
[0146] After centrifugation, the supernatant was applied to AKTA
prime (GE Healthcare) to purify the antibody. In this regard, 100
mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA
Prime equipped with a Protein A column (GE healthcare, 17-0405-03),
followed by elution with an IgG elution buffer (Thermo Scientific,
21004). The buffer was exchanged with PBS to purify a chimeric
antibody AbF46 (hereinafter referred to as "chAbF46").
[0147] 1.3. Construction of Humanized Antibody huAbF46 from
Chimeric Antibody chAbF46
[0148] 1.3.1. Heavy Chain Humanization
[0149] To design two domains H1-heavy and H3-heavy, human germline
genes which share the highest identity/homology with the VH gene of
the mouse antibody AbF46 purified in Reference Example 1.2 were
analyzed. An Ig BLAST (NCBI) result revealed that VH3-71 has an
identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2,
and CDR-H3 of the mouse antibody AbF46 were defined according to
Kabat numbering. A design was made to introduce the CDR of the
mouse antibody AbF46 into the framework of VH3-71. Hereupon, back
mutations to the amino acid sequence of the mouse AbF46 were
conducted at positions 30 (S.fwdarw.T), 48 (V.fwdarw.L), 73
(D.fwdarw.N), and 78 (T.fwdarw.L). Then, H1 was further mutated at
positions 83 (R.fwdarw.K) and 84 (A.fwdarw.T) to finally establish
H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).
[0150] For use in designing H4-heavy, human antibody frameworks
were analyzed by a BLAST search. The result revealed that the VH3
subtype, known to be most stable, is very similar in framework and
sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of
the mouse antibody AbF46 were defined according to Kabat numbering
and introduced into the VH3 subtype to construct H4-heavy (SEQ ID
NO: 42).
[0151] 1.3.2. Light Chain Humanization
[0152] To design two domains H1-light (SEQ ID NO: 43) and H2-light
(SEQ ID NO: 44), human germline genes which share the highest
identity/homology with the VH gene of the mouse antibody AbF46 were
analyzed. An Ig BLAST search result revealed that VK4-1 has a
identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2,
and CDR-L3 of the mouse antibody AbF46 were defined according to
Kabat numbering. A design was made to introduce the CDR of the
mouse antibody AbF46 into the framework of VK4-1. Hereupon, back
mutations to the amino acid sequence of the mouse AbF46 were
conducted at positions 36 (Y.fwdarw.H), 46 (L.fwdarw.M), and 49
(Y.fwdarw.I). Only one back mutation was conducted at position 49
(Y.fwdarw.I) on H2-light.
[0153] To design H3-light (SEQ ID NO: 45), human germline genes
which share the highest identity/homology with the VL gene of the
mouse antibody AbF46 were analyzed by a search for BLAST. As a
result, VK2-40 was selected. VL and VK2-40 of the mouse antibody
AbF46 were found to have a identity/homology of 61% at an amino
acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were
defined according to Kabat numbering and introduced into the
framework of VK4-1. Back mutations were conducted at positions 36
(Y.fwdarw.H), 46 (L.fwdarw.M), and 49 (Y.fwdarw.I) on H3-light.
[0154] For use in designing H4-light (SEQ ID NO: 46), human
antibody frameworks were analyzed. A Blast search revealed that the
Vk1 subtype, known to be the most stable, is very similar in
framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2,
and CDR-L3 of the mouse antibody AbF46 were defined according to
Kabat numbering and introduced into the Vk1 subtype. Hereupon, back
mutations were conducted at positions 36 (Y.fwdarw.H), 46
(L.fwdarw.M), and 49 (Y.fwdarw.1) on H4-light.
[0155] Thereafter, DNA fragments having the heavy chain nucleotide
sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48,
H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain
nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO:
51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested
with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into
a pOptiVEC.TM.-TOPO TA Cloning Kit enclosed in an OptiCHO.TM.
Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a
pcDNA.TM. 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively,
so as to construct recombinant vectors for expressing a humanized
antibody.
[0156] Each of the constructed vectors was amplified using Qiagen
Maxiprep kit (Cat no. 12662), and a transient expression was
performed using Freestyle.TM. MAX 293 Expression System
(invitrogen). 293 F cells were used for the expression and cultured
in FreeStyle.TM. 293 Expression Medium in a suspension culture
manner. At one day before the transient expression, the cells were
provided in the concentration of 5.times.10.sup.5 cells/ml, and
after 24 hours, when the cell number reached to 1.times.10.sup.6
cells/ml, the transient expression was performed. A transfection
was performed by a liposomal reagent method using Freestyle.TM. MAX
reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided
in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and
mixed with 2 ml of OptiPro.TM. SFM (invitrogen) (A), and in another
15 ml tube, 100 ul (microliter) of Freestyle.TM. MAX reagent and 2
ml of OptiPro.TM. SFM were mixed (B), followed by mixing (A) and
(B) and incubating for 15 minutes. The obtained mixture was slowly
mixed with the cells provided one day before the transient
expression. After completing the transfection, the cells were
incubated in 130 rpm incubator for 5 days under the conditions of
37.degree. C., 80% humidity, and 8% CO.sub.2.
[0157] After centrifugation, the supernatant was applied to AKTA
prime (GE Healthcare) to purify the antibody. In this regard, 100
mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA
Prime equipped with a Protein A column (GE healthcare, 17-0405-03),
followed by elution with an IgG elution buffer (Thermo Scientific,
21004). The buffer was exchanged with PBS to purify a humanized
antibody AbF46 (hereinafter referred to as "huAbF46"). The
humanized antibody huAbF46 used in the following examples comprised
a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO:
46).
[0158] 1.4. Construction of scFV Library of huAbF46 Antibody
[0159] For use in constructing an scFv of the huAbF46 antibody from
the heavy and light chain variable regions of the huAbF46 antibody,
a gene was designed to have the structure of "VH-linker-VL" for
each of the heavy and the light chain variable region, with the
linker having the amino acid sequence "GLGGLGGGGSGGGGSGGSSGVGS"
(SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding
the designed scFv of huAbF46 was synthesized in Bioneer and an
expression vector for the polynucleotide had the nucleotide
sequence of SEQ ID NO: 56.
[0160] After expression, the product was found to exhibit
specificity to c-Met.
[0161] 1.5. Construction of Library Genes for Affinity
Maturation
[0162] 1.5.1. Selection of Target CDRs and Synthesis of Primers
[0163] The affinity maturation of huAbF46 was achieved. First, six
complementary determining regions (CDRs) were defined according to
Kabat numbering. The CDRs are given in Table 1, below.
TABLE-US-00007 TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID
NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG(SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ
ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS
(SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)
[0164] For use in the introduction of random sequences into the
CDRs of the antibody, primers were designed as follows.
Conventionally, N codons were utilized to introduce bases at the
same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of
mutation. In this experiment, the introduction of random bases into
the CDRs of huAbF46 was conducted in such a manner that, of the
three nucleotides per codon in the wild-type polynucleotide
encoding each CDR, the first and second nucleotides conserved over
85% of the entire sequence while the other three nucleotides were
introduced at the same percentage (each 5%) and that the same
possibility was imparted to the third nucleotide (33% G, 33% C, 33%
T).
[0165] 1.5.2. Construction of a Library of huAbF46 Antibodies and
Affinity for c-Met
[0166] The construction of antibody gene libraries through the
introduction of random sequences was carried out using the primers
synthesized in the same manner as in Reference Example 1.5.1. Two
PCR products were obtained using a polynucleotide covering the scFV
of huAbF46 as a template, and were subjected to overlap extension
PCR to give scFv library genes for huAbF46 antibodies in which only
desired CDRs were mutated. Libraries targeting each of the six CDRs
prepared from the scFV library genes were constructed.
[0167] The affinity for c-Met of each library was compared to that
of the wildtype. Most libraries were lower in affinity for c-Met,
compared to the wild-type. The affinity for c-Met was retained in
some mutants.
[0168] 1.6. Selection of Antibody with Improved Affinity from
Libraries
[0169] After maturation of the affinity of the constructed
libraries for c-Met, the nucleotide sequence of scFv from each
clone was analyzed. The nucleotide sequences thus obtained are
summarized in Table 2 and were converted into IgG forms. Four
antibodies which were respectively produced from clones L3-1, L3-2,
L3-3, and L3-5 were used in the subsequent experiments.
TABLE-US-00008 TABLE 2 Library Clone constructed CDR Sequence H11-4
CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23)
YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID
NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY
(SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1
KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA
(SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12
CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1
KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO:
34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ
ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3
GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15)
L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ
ID NO: 37)
[0170] 1.7. Conversion of Selected Antibodies into IgG
[0171] Respective polynucleotides encoding heavy chains of the four
selected antibodies were designed to have the structure of
"EcoRI-signal sequence-VH-NheI-CH-XhoI" (SEQ ID NO: 38). The heavy
chains of huAbF46 antibodies were used as they were because their
amino acids were not changed during affinity maturation. In the
case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57)
was employed instead of the hinge of human IgG1. Genes were also
designed to have the structure of "EcoRI-signal
sequence-VL-BsiWI-CL-XhoI" for the light chain. Polypeptides
encoding light chain variable regions of the four antibodies which
were selected after the affinity maturation were synthesized in
Bioneer. Then, a DNA fragment having the heavy chain nucleotide
sequence (SEQ ID NO: 38) and DNA fragments having the light chain
nucleotide sequences (DNA fragment comprising L3-1-derived CDR-L3:
SEQ ID NO: 58, DNA fragment comprising L3-2-derived CDR-L3: SEQ ID
NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60,
and DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61)
were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S)
before cloning into a pOptiVEC.TM.-TOPO TA Cloning Kit enclosed in
an OptiCHO.TM. Antibody Express Kit (Cat no. 12762-019, Invitrogen)
and a pcDNA.TM. 3.3-TOPO TA Cloning Kit (Cat no. 8300-01),
respectively, so as to construct recombinant vectors for expressing
affinity-matured antibodies.
[0172] Each of the constructed vectors was amplified using Qiagen
Maxiprep kit (Cat no. 12662), and a transient expression was
performed using Freestyle.TM. MAX 293 Expression System
(invitrogen). 293 F cells were used for the expression and cultured
in FreeStyle.TM. 293 Expression Medium in a suspension culture
manner. At one day before the transient expression, the cells were
provided in the concentration of 5.times.10.sup.5 cells/ml, and
after 24 hours, when the cell number reached to 1.times.10.sup.6
cells/ml, the transient expression was performed. A transfection
was performed by a liposomal reagent method using Freestyle.TM. MAX
reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided
in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and
mixed with 2 ml of OptiPro.TM. SFM (invitrogen) (A), and in another
15 ml tube, 100 ul (microliter) of Freestyle.TM. MAX reagent and 2
ml of OptiPro.TM. SFM were mixed (B), followed by mixing (A) and
(B) and incubating for 15 minutes. The obtained mixture was slowly
mixed with the cells provided one day before the transient
expression. After completing the transfection, the cells were
incubated in 130 rpm incubator for 5 days under the conditions of
37.degree. C., 80% humidity, and 8% CO.sub.2.
[0173] After centrifugation, the supernatant was applied to AKTA
prime (GE Healthcare) to purify the antibody. In this regard, 100
mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA
Prime equipped with a Protein A column (GE healthcare, 17-0405-03),
followed by elution with an IgG elution buffer (Thermo Scientific,
21004). The buffer was exchanged with PBS to purify four
affinity-matured antibodies (hereinafter referred to as
"huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin),
huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),"
respectively).
[0174] 1.8. Construction of Constant Region- and/or Hinge
Region-Substituted huAbF46-H4-A1
[0175] Among the four antibodies selected in Reference Example 1.7,
huAbF46-H4-A1 was found to be the highest in affinity for c-Met and
the lowest in Akt phosphorylation and c-Met degradation degree. In
the antibody, the hinge region, or the constant region and the
hinge region, were substituted.
[0176] The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy
chain comprising the heavy chain variable region of huAbF46-H4-A1,
U6-HC7 hinge, and the constant region of human IgG1 constant
region, and a light chain comprising the light chain variable
region of huAbF46-H4-A1 and human kappa constant region. The
antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain
comprising a heavy chain variable region, a human IgG2 hinge
region, and a human IgG1 constant region, and a light chain
comprising the light chain variable region of huAbF46-H4-A1 and a
human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc)
was composed of the heavy chain variable region of huAbF46-H4-A1, a
human IgG2 hinge region, and a human IgG2 constant region, and a
light chain comprising the light variable region of huAbF46-H4-A1
and a human kappa constant region. Hereupon, the histidine residue
at position 36 on the human kappa constant region of the light
chain was changed to tyrosine in all of the three antibodies to
increase antibody production.
[0177] For use in constructing the three antibodies, a
polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO:
62) composed of the heavy chain variable region of huAbF46-H4-A1, a
U6-HC7 hinge region, and a human IgG1 constant region, a
polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO:
64) composed of the heavy chain variable region of huAbF46-H4-A1, a
human IgG2 hinge region, and a human IgG1 region, a polynucleotide
(SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of
the heavy chain variable region of huAbF46-H4-A1, a human IgG2
region, and a human IgG2 constant region, and a polynucleotide (SEQ
ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the
light chain variable region of huAbF46-H4-A1, with a tyrosine
residue instead of histidine at position 36, and a human kappa
constant region were synthesized in Bioneer. Then, the DNA
fragments having heavy chain nucleotide sequences were inserted
into a pOptiVEC.TM.-TOPO TA Cloning Kit enclosed in an OptiCHO.TM.
Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA
fragments having light chain nucleotide sequences were inserted
into a pcDNA.TM. 3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to
construct vectors for expressing the antibodies.
[0178] Each of the constructed vectors was amplified using Qiagen
Maxiprep kit (Cat no. 12662), and a transient expression was
performed using Freestyle.TM. MAX 293 Expression System
(invitrogen). 293 F cells were used for the expression and cultured
in FreeStyle.TM. 293 Expression Medium in a suspension culture
manner. At one day before the transient expression, the cells were
provided in the concentration of 5.times.10.sup.5 cells/ml, and
after 24 hours, when the cell number reached to 1.times.10.sup.6
cells/ml, the transient expression was performed. A transfection
was performed by a liposomal reagent method using Freestyle.TM. MAX
reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided
in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and
mixed with 2 ml of OptiPro.TM. SFM (invitrogen) (A), and in another
15 ml tube, 100 ul (microliter) of Freestyle.TM. MAX reagent and 2
ml of OptiPro.TM. SFM were mixed (B), followed by mixing (A) and
(B) and incubating for 15 minutes. The obtained mixture was slowly
mixed with the cells provided one day before the transient
expression. After completing the transfection, the cells were
incubated in 130 rpm incubator for 5 days under the conditions of
37.degree. C., 80% humidity, and 8% CO.sub.2.
[0179] After centrifugation, the supernatant was applied to AKTA
prime (GE Healthcare) to purify the antibody. In this regard, 100
mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA
Prime equipped with a Protein A column (GE healthcare, 17-0405-03),
followed by elution with IgG elution buffer (Thermo Scientific,
21004). The buffer was exchanged with PBS to finally purify three
antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and
huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1
(U6-HC7) was selected for the following examples, and referred as
anti-c-Met antibody L3-1Y.
Reference Example 2
Preparation of a Bispecific Antibody
[0180] The anti c-Met antibodies manufactured in Reference Example
1 were fused to a linker by coupling the C-terminal of their heavy
chain therewith. Thereafter, an Ig2 domain (SEQ ID NO: 114), that
is, amino acids from 129.sup.th to 229.sup.th among the amino acids
constituting VEGF receptor 1 (P17948.2; SEQ ID NO: 113), was
sequentially fused at the terminal of the linker to manufacture
antibodies capable of binding to c-Met and VEGF at the same time
(see FIG. 1).
[0181] Among 1338 amino acids constituting VEGF receptor 1
(P17948.2; SEQ ID NO: 113), a gene sequence encoding 101 amino
acids from 129.sup.th to 229.sup.th position constituting the Ig2
domain which has been known to be most important for VEGF-binding
was secured from NCBI database.
TABLE-US-00009 Ig2 domain (VIG2) amino acid sequence (SEQ ID NO:
114): SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTI Ig2 domain
(VIG2) nucleotide sequence (SEQ ID NO: 115):
AGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAA
TTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTAC
GTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTG
ATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCA
TATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAAC
AGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACC AATACAATC
[0182] In order to couple the heavy chain of the c-Met antibody
manufactured in the above and the Ig2 domain (VIG2), three types of
linkers, `GGGGS`(G4S) (SEQ ID NO: 116), `GGGGSGGGGS`((G4S)2) (SEQ
ID NO: 117), or `GGGGSGGGGSGGGGSGGGGS`((G4S)4) (SEQ ID NO: 118),
out of the structures where GGGGS are repeated were designed, they
were placed between the c-Met antibody and the Ig2 domain (VIG2) of
VEGF receptor 1, and the synthesis of a gene in which a stop codon
(TGA) is inserted into the end of the designed final gene was
requested to Bionia. The thus synthesized gene was inserted into
pOptivec vector (Invitrogen) using an EcoRI/XhoI cloning site to
produce a heavy chain expression vector. The vector used in the
manufacture of L3-1Y was also used as a light chain expression
vector.
[0183] Each of the thus constructed vectors was amplified using
Qiagen Maxiprep kit (Cat no. 12662), and the vector including the
heavy chain and the vector containing the light chain were
transfected at the ratio of 4:1 into 293T cells
(2.5.times.10.sup.7) to which 360 .mu.l of 2M CaCl.sub.2 was added.
Thereafter, the transfected cells were cultured in a DMEM medium
containing 10% FBS at 37.degree. C. in 5% CO.sub.2 conditions for 5
hours, and then cultured in an FBS-free DMEM medium at 37.degree.
C. in 5% CO.sub.2 conditions for 48 hours.
[0184] The cultured cells were centrifuged to obtain 100 ml of each
supernatant, which was purified using AKTA Prime (GE healthcare).
Protein A column (GE healthcare, 17-0405-03) was placed in the AKTA
Prime, and the cultured solution was flowed at a flow rate of 5
ml/min and was eluted with IgG elution buffer (Thermo Scientific,
21004). The buffer was exchanged with a PBS buffer, and thus
antibodies capable of binding to cMet and VEGF at the same time
were finally purified.
[0185] The thus prepared antibodies can be summarized as
follows:
TABLE-US-00010 TABLE 3 Heavy Constant VEGF-binding Name Chain Hinge
Region Linker Light Chain fragment Bispecific MV10A SEQ ID U6-HC7
IgG1 (G4S)2 SEQ ID VIG2 antibody NO: 62 (SEQ ID NO: 70 (SEQ ID NO:
114) NO: 101) MV10AY SEQ ID U6-HC7 IgG1 (G4S)2 SEQ ID VIG2 NO: 62
(SEQ ID NO: 68 (SEQ ID NO: 114) NO: 101) MV10AY SEQ ID U3 HC9 IgG1
(G4S)2 SEQ ID VIG2 U3 NO: 64 (SEQ ID NO: 68 (SEQ ID NO: 114) HC9/
NO: 102) IgG1 MV10AY SEQ ID U3 HC9 IgG2 (G4S)2 SEQ ID VIG2 U3 NO:
66 (SEQ ID NO: 68 (SEQ ID NO: 114) HC9/ NO: 102) IgG2
[0186] Among the prepared bispecific antibodies, MV10AY was
selected for the following examples, and referred as "VIG2".
Example 1
Confirmation of the Effect of Combined Use of Erlotinib and
Anti-cMet Antibody
[0187] The effect of co-administration of an anti-c-Met antibody
L3-1Y and erlotinib was confirmed in HCC827 human lung cancer cell
line.
[0188] In a 96 well plate, diluted HCC827 human lung cancer cell
line (CRL-2868, ATCC) was seeded at 10,000 cells/well in a RPMI1640
medium (GIBCO) containing 10% FBS, and then, incubated at
37.degree. C. overnight. On the next day, the antibody (RPMI1640
medium) was diluted to 0.008 .mu.g/ml, and the cultured cells were
treated therewith at 100 .mu.l (microliter) per well. For
co-administration, the cells were treated with 80 nM of an EGFR
antagonist erlotinib (Roche), together with the antibody. After 3
days, the number of cells was measured using a CCK-8 reagent
(Dojindo laboratory). As the antibody, the c-Met antibody L3-1Y
manufactured in Reference Example 1 was used to compare the effect
of combined use.
[0189] The results are shown in FIG. 1. It was confirmed that the
growth of cell line was effectively inhibited when erlotinib and
antibody L3-1Y are co-administered, compared to single
administrations thereof (see FIG. 1), indicating that simultaneous
inhibition of EGFR and c-Met may effectively enhance anticancer
activity.
Example 2
Preparation of Erlotinib Resistant Cell Line
[0190] HCC827 human lung cancer cell line was in vitro treated with
an EGFR antagonist erlotinib for a given period to establish HCC827
ER(HCC827 erlotinib-resistant) cell line. More specifically, HCC827
human lung cancer cell line (CRL-2868, ATCC) was in vitro treated
with an EGFR antagonist erlotinib (Roche) in an amount of 5 nM to
10 nM for 5 months or more, to construct Erlotinib resistant cell
(ER cell) clone.
Example 3
Confirmation of c-Met gDNA Copy Number in Erlotinib Resistant Cell
Line
[0191] Various clones of the erlotinib resistant cell line were
selected, and c-Met copy number was confirmed through gDNA
real-time PCR.
[0192] The relative copy number of c-Met genes was measured using
7900HT Fast real-time PCR systems (Applied Biosystems, CA, USA),
and a SYBR Green PCR master mix kit (Applied Biosystems, CA, USA).
To determine c-Met gene copy number in cells, a relative value to a
serially diluted reference samples was obtained to calculate copy
number. Wherein, the used primers are as follows.
TABLE-US-00011 c-Met: (forward: SEQ ID NO: 109)
5'-ACCTGCCAGCGACATGTCTT-3' (reverse: SEQ ID NO: 110)
5'-GACACTGGCTGGGCTCTTCTATC-3' Actin: (forward: SEQ ID NO: 111)
5'-TCACCCACACTGTGCCCATCTACGA-3' (reverse: SEQ ID NO: 112)
5'-TCGGTGAGGATCTTCATGAGGTA-3'
[0193] The results are shown in FIG. 2.
[0194] As shown in FIG. 2, it was confirmed that c-Met genes were
amplified 3 times or more in all erlotinib resistant cell lines,
particularly c-Met gene level over 4 times in clones 10 and 15,
indicating that by continuous exposure to an EGFT antagonist
(inhibitor), c-Met level may increase, and thereby, resistance may
occur.
Example 4
Confirmation of Erlotinib Resistance
[0195] The clones 10 and 15 exhibiting high c-Met amplification
degrees in Example 3 (respectively named as HCC827 ER #10 and
HCC827 ER #15) were selected to confirm erlotinib resistance
occurrence.
[0196] To confirm erlotinib resistance occurrence, the HCC827 and
HCC827 ER #15 cell lines were respectively treated with erlotinib
at various concentrations for 120 hours, and then, the number of
cells was measured using a CCK-8 reagent (Dojindo laboratory), and
the results are shown in FIG. 3.
[0197] As shown in FIG. 3, it was confirmed that resistance
occurred to other EGFR antagonist Afatinib (BIBW2992; selleckchem)
as well as to erlotinib. It means a possibility that resistance may
occur to other EGFR antagonists beside them.
Example 5
Confirmation of Combined Use of Erlotinib and Anti-c-Met L3-1Y in
Erlotinib Resistant Cell Line
[0198] In the HCC827 ER #15, of which resistance to erlotinib was
confirmed in Example 4, the effects of single treatments and
combined treatment of erlotinib and the anti-c-Met antibody L3-1Y
manufactured in the Reference Example were tested.
[0199] In a 96 well plate, diluted HCC827 ER #15 was seeded at 5000
cells/well in a RPMI164 medium (GIBCO) containing 10% FBS, and
then, incubated at 37.degree. C. overnight. On the next day, the
medium was replaced with a RPMI1640 medium containing 1% FBS
(GIBCO), and then, the antibody was serially diluted from 100
ug/ml, and the cells were treated therewith at 100 ul. For
co-administration, the cells were treated with 10 nM or 100 nM
erlotinib, together with the antibody. After 5 days, the number of
cells was measured using a CCK-8 reagent (Dojindo laboratory). As
the antibody, the anti-c-Met antibody L3-1Y manufactured in the
Reference Example 1 and VIG2, and ref(anti-c-Met antibody, Eli
lilly and Company), Rituximab (IDEK), and Cetuximab (BMS) were
used, and the effects were compared.
[0200] The results are shown in FIGS. 4a to 4d. It was confirmed
that in the case of co-administration of erlotinib and antibody
L3-1Y or VIG2, the growth of cell line may be effectively inhibited
even at a very low concentration, compared to single administration
of each of them in HCC827 ER #15 cell line (see FIGS. 4a and 4b).
When another c-Met antibody ref (Eli lilly) was used, similar
effects were also observed (see FIG. 4c). Compared to the case of
using ref antibody, when antibody L3-1Y or VIG2 was co-administered
with erlotinib, excellent synergistic effect was exhibited. As
confirmed in FIGS. 4a and 4b, in the case of co-administration of
antibody L3-1Y or VIG2 and erlotinib, maximum efficiency was
already exhibited below a L3-1Y or VIG2 dose of 0.1 .mu.g/ml,
indicating that simultaneous inhibition of EGFR and c-Met in cell
line in which EGFR antagonist resistance occurs may be effective
for anticancer activity and overcoming of resistance.
[0201] And, in the case wherein Rituximab or Cetuximab, targeting
other materials instead of c-Met antibody, is co-administered with
erlotinib, synergistic effect was not observed (FIG. 4d). It means
that the effects of co-administration of erlotinib and antibody
L3-1Y results from c-Met specific inhibition of L3-1Y.
[0202] Meanwhile, even when a test was conducted again while fixing
the concentration of the drugs used for co-administration, the
identical effects could be confirmed. The cell viability of HCC827
ER#15 is shown in FIG. 5 when the antibodies and erlotinib were
co-administered while fixing the concentration of the antibodies at
0.14 .mu.g/ml, and the concentration of erlotinib at 10 nM. As
shown in FIG. 5, synergistic effects due to co-administration of
erlotinib and c-Met antibodies were confirmed again, while
synergistic effects were not observed when materials other than
c-Met antibodies, such as Rituximab or Cetuximab were
co-administered with erlotinib. These results are of great
significance in that the used drug concentrations are within a
level reachable in the body of a practical patient (e.g., in the
case of commercialized Cetuximab, 1-5 ug/ml Cmax).
Example 6
Confirmation of Cbl and LRIG1 Protein Expression Amount in
Erlotinib Resistant Cell Lines
[0203] In HCC827, HCC827 ER #10, and HCC827 ER #15 cells, p-c-Met,
c-Met, EGFR, Cbl, LRIG1, and GAPDH expression amounts were
respectively analyzed by immunoblotting using each antibody.
Wherein, antibodies to p-c-Met, c-Met, EGFR, Cbl, and GAPDH (14C10)
were purchased from Cell Signaling Technology, Inc., and antibody
to LRIG1 was purchased from AbCam.
[0204] The results are shown in FIG. 6. As shown in FIG. 6, it was
confirmed that in erlotinib resistant cell lines, c-Met expression
amount was relatively high, and EGFR expression amount was
relatively low, compared to non-resistant HCC827 cell line. With
regard to the expression amounts of Cbl and LRIG1, important
regulators participating in degradation of RKT such as c-Met and
EGFR or inhibition of the functions, the Cbl expression amount in
HCC827 ER #15 in which the effect of combined use was confirmed was
significantly decreased, but the LRIG1 expression amount was not
significantly decreased. Namely, it can be seen that L3-1Y or VIG2
antibody may achieve excellent effects of combined use even for
cancers in which Cbl expression amount is low.
[0205] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0206] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0207] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
11815PRTArtificial SequenceSynthetic (heavy chain CDR1 of AbF46)
1Asp Tyr Tyr Met Ser 1 5 219PRTArtificial SequenceSynthetic (heavy
chain CDR2 of AbF46) 2Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr
Glu Tyr Ser Ala Ser 1 5 10 15 Val Lys Gly 36PRTArtificial
SequenceSynthetic (heavy chain CDR3 of AbF46) 3Asp Asn Trp Phe Ala
Tyr 1 5 46PRTArtificial SequenceSynthetic (heavy chain CDR1 of
c-Met antibody) 4Xaa Xaa Tyr Tyr Met Ser 1 5 58PRTArtificial
SequenceSynthetic (heavy chain CDR2 of c-Met antibody) 5Arg Asn Xaa
Xaa Asn Gly Xaa Thr 1 5 66PRTArtificial SequenceSynthetic (heavy
chain CDR3 of c-Met antibody) 6Asp Asn Trp Leu Xaa Tyr 1 5
717PRTArtificial SequenceSynthetic (light chain CDR1 of c-Met
antibody) 7Lys Ser Ser Xaa Ser Leu Leu Ala Xaa Gly Asn Xaa Xaa Asn
Tyr Leu 1 5 10 15 Ala 87PRTArtificial SequenceSynthetic (light
chain CDR2 of c-Met antibody) 8Trp Xaa Ser Xaa Arg Val Xaa 1 5
99PRTArtificial SequenceSynthetic (light chain CDR3 of c-Met
antibody) 9Xaa Gln Ser Tyr Ser Xaa Pro Xaa Thr 1 5
1017PRTArtificial SequenceSynthetic (light chain CDR1 of AbF46)
10Lys Ser Ser Gln Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu 1
5 10 15 Ala 117PRTArtificial SequenceSynthetic (light chain CDR2 of
AbF46) 11Trp Ala Ser Thr Arg Val Ser 1 5 129PRTArtificial
SequenceSynthetic (light chain CDR3 of AbF46) 12Gln Gln Ser Tyr Ser
Ala Pro Leu Thr 1 5 139PRTArtificial SequenceSynthetic (CDR-L3
derived from L3-1 clone) 13Gln Gln Ser Tyr Ser Arg Pro Tyr Thr 1 5
149PRTArtificial SequenceSynthetic (CDR-L3 derived from L3-2 clone)
14Gly Gln Ser Tyr Ser Arg Pro Leu Thr 1 5 159PRTArtificial
SequenceSynthetic (CDR-L3 derived from L3-3 clone) 15Ala Gln Ser
Tyr Ser His Pro Phe Ser 1 5 169PRTArtificial SequenceSynthetic
(CDR-L3 derived from L3-5 clone) 16Gln Gln Ser Tyr Ser Arg Pro Phe
Thr 1 5 17117PRTArtificial SequenceSynthetic (heavy chain variable
region of anti c-Met humanized antibody(huAbF46-H4)) 17Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr
Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe
Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
18114PRTArtificial SequenceSynthetic (light chain variable region
of anti c-Met humanized antibody(huAbF46-H4)) 18Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly
Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40
45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val
50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg 19114PRTArtificial
SequenceSynthetic (light chain variable region of anti c-Met
humanized antibody(huAbF46-H4)) 19Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn
Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro
Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gly Gln 85 90 95 Ser Tyr Ser Arg Pro Leu Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys Arg 20114PRTArtificial
SequenceSynthetic (light chain variable region of anti c-Met
humanized antibody(huAbF46-H4)) 20Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn
Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro
Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Ala Gln 85 90 95 Ser Tyr Ser His Pro Phe Ser Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys Arg 21114PRTArtificial
SequenceSynthetic (light chain variable region of anti c-Met
humanized antibody(huAbF46-H4)) 21Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn
Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro
Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln 85 90 95 Ser Tyr Ser Arg Pro Phe Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys Arg 226PRTArtificial
SequenceSynthetic (CDR-H1 derived from H11-4 clone) 22Pro Glu Tyr
Tyr Met Ser 1 5 236PRTArtificial SequenceSynthetic (CDR-H1 derived
from YC151 clone) 23Pro Asp Tyr Tyr Met Ser 1 5 246PRTArtificial
SequenceSynthetic (CDR-H1 derived from YC193 clone) 24Ser Asp Tyr
Tyr Met Ser 1 5 258PRTArtificial SequenceSynthetic (CDR-H2 derived
from YC244 clone) 25Arg Asn Asn Ala Asn Gly Asn Thr 1 5
268PRTArtificial SequenceSynthetic (CDR-H2 derived from YC321
clone) 26Arg Asn Lys Val Asn Gly Tyr Thr 1 5 276PRTArtificial
SequenceSynthetic (CDR-H3 derived from YC354 clone) 27Asp Asn Trp
Leu Ser Tyr 1 5 286PRTArtificial SequenceSynthetic (CDR-H3 derived
from YC374 clone) 28Asp Asn Trp Leu Thr Tyr 1 5 2917PRTArtificial
SequenceSynthetic (CDR-L1 derived from L1-1 clone) 29Lys Ser Ser
His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu 1 5 10 15 Ala
3017PRTArtificial SequenceSynthetic (CDR-L1 derived from L1-3
clone) 30Lys Ser Ser Arg Ser Leu Leu Ser Ser Gly Asn His Lys Asn
Tyr Leu 1 5 10 15 Ala 3117PRTArtificial SequenceSynthetic (CDR-L1
derived from L1-4 clone) 31Lys Ser Ser Lys Ser Leu Leu Ala Ser Gly
Asn Gln Asn Asn Tyr Leu 1 5 10 15 Ala 3217PRTArtificial
SequenceSynthetic (CDR-L1 derived from L1-12 clone) 32Lys Ser Ser
Arg Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu 1 5 10 15 Ala
3317PRTArtificial SequenceSynthetic (CDR-L1 derived from L1-22
clone) 33Lys Ser Ser His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn
Tyr Leu 1 5 10 15 Ala 347PRTArtificial SequenceSynthetic (CDR-L2
derived from L2-9 clone) 34Trp Ala Ser Lys Arg Val Ser 1 5
357PRTArtificial SequenceSynthetic (CDR-L2 derived from L2-12
clone) 35Trp Gly Ser Thr Arg Val Ser 1 5 367PRTArtificial
SequenceSynthetic (CDR-L2 derived from L2-16 clone) 36Trp Gly Ser
Thr Arg Val Pro 1 5 379PRTArtificial SequenceSynthetic (CDR-L3
derived from L3-32 clone) 37Gln Gln Ser Tyr Ser Lys Pro Phe Thr 1 5
381416DNAArtificial SequenceSynthetic (nucleotide sequence of heavy
chain of chAbF46) 38gaattcgccg ccaccatgga atggagctgg gtttttctcg
taacactttt aaatggtatc 60cagtgtgagg tgaagctggt ggagtctgga ggaggcttgg
tacagcctgg gggttctctg 120agactctcct gtgcaacttc tgggttcacc
ttcactgatt actacatgag ctgggtccgc 180cagcctccag gaaaggcact
tgagtggttg ggttttatta gaaacaaagc taatggttac 240acaacagagt
acagtgcatc tgtgaagggt cggttcacca tctccagaga taattcccaa
300agcatcctct atcttcaaat ggacaccctg agagctgagg acagtgccac
ttattactgt 360gcaagagata actggtttgc ttactggggc caagggactc
tggtcactgt ctctgcagct 420agcaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 480acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
600ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 660atctgcaacg tgaatcacaa gcccagcaac accaaggtgg
acaagaaagt tgagcccaaa 720tcttgtgaca aaactcacac atgcccaccg
tgcccagcac ctgaactcct ggggggaccg 780tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 840gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
900gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 960acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 1020tacaagtgca aggtctccaa caaagccctc
ccagccccca tcgagaaaac catctccaaa 1080gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc
1200gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc
tcccgtgctg 1260gactccgacg gctccttctt cctctacagc aagctcaccg
tggacaagag caggtggcag 1320caggggaacg tcttctcatg ctccgtgatg
catgaggctc tgcacaacca ctacacgcag 1380aagagcctct ccctgtctcc
gggtaaatga ctcgag 141639759DNAArtificial SequenceSynthetic
(nucleotide sequence of light chain of chAbF46) 39gaattcacta
gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60ctgctgctat
cggtatctgg tacctgtgga gacattttga tgacccagtc tccatcctcc
120ctgactgtgt cagcaggaga gaaggtcact atgagctgca agtccagtca
gagtctttta 180gctagtggca accaaaataa ctacttggcc tggcaccagc
agaaaccagg acgatctcct 240aaaatgctga taatttgggc atccactagg
gtatctggag tccctgatcg cttcataggc 300agtggatctg ggacggattt
cactctgacc atcaacagtg tgcaggctga agatctggct 360gtttattact
gtcagcagtc ctacagcgct ccgctcacgt tcggtgctgg gaccaagctg
420gagctgaaac gtacggtggc tgcaccatct gtcttcatct tcccgccatc
tgatgagcag 480ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata
acttctatcc cagagaggcc 540aaagtacagt ggaaggtgga taacgccctc
caatcgggta actcccagga gagtgtcaca 600gagcaggaca gcaaggacag
cacctacagc ctcagcagca ccctgacgct gagcaaagca 660gactacgaga
aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc
720gtcacaaaga gcttcaacag gggagagtgt tgactcgag 75940447PRTArtificial
SequenceSynthetic (amino acid sequence of H1-heavy) 40Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr
Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe
Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155
160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405
410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 445 41447PRTArtificial SequenceSynthetic (amino
acid sequence of H3-heavy) 41Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile
Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195
200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
440 445 42447PRTArtificial SequenceSynthetic (amino acid sequence
of H4-heavy) 42Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asn Lys Ala
Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr
Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230
235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355
360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 43220PRTArtificial
SequenceSynthetic (amino acid sequence of H1-light) 43Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25
30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Gln
35 40 45 Pro Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser
Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155
160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 215 220 44220PRTArtificial SequenceSynthetic (amino
acid sequence of H2-light) 44Asp Ile Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn
Tyr Leu Ala Trp His Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln
Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70
75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln
Gln 85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys
Leu Glu Leu 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195
200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220
45220PRTArtificial SequenceSynthetic (amino acid sequence of
H3-light) 45Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Ile Trp
Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr
Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115
120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 46219PRTArtificial
SequenceSynthetic (amino acid sequence of H4-light) 46Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25
30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys
35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser
Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155
160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu 210 215 471350DNAArtificial SequenceSynthetic (nucleotide
sequence of H1-heavy) 47gaggtgcagc tggtggagtc tgggggaggc ttggtccagc
ctggagggtc cctgagactc 60tcctgtgcag cctctggatt caccttcact gactactaca
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg gttgggcttt
attagaaaca aagctaacgg ttacaccaca 180gaatacagtg cgtctgtgaa
aggcagattc accatctcaa gagataattc aaagaactca 240ctgtatctgc
aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgctaga
300gataactggt ttgcttactg gggtcaagga accctggtca ccgtctcctc
ggctagcacc 360aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 420gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 540tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 660gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
900cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1200gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg
gcagcagggg 1260aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1320ctctccctgt ctccgggtaa atgactcgag
1350481350DNAArtificial SequenceSynthetic (nucleotide sequence of
H3-heavy) 48gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcact gactactaca tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg gttgggcttt attagaaaca
aagctaacgg ttacaccaca 180gaatacagtg cgtctgtgaa aggcagattc
accatctcaa gagataattc aaagaactca 240ctgtatctgc aaatgaacag
cctgcgtgct gaggacacgg ccgtgtatta ctgtgctaga 300gataactggt
ttgcttactg gggtcaagga accctggtca ccgtctcctc ggctagcacc
360aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 420gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 540tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc
acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt
660gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1260aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1320ctctccctgt ctccgggtaa atgactcgag
1350491350DNAArtificial SequenceSynthetic (nucleotide sequence of
H4-heavy) 49gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc
actccgtttg 60tcctgtgcag cttctggctt caccttcact gattactaca tgagctgggt
gcgtcaggcc 120ccgggtaagg gcctggaatg gttgggtttt attagaaaca
aagctaatgg ttacacaaca 180gagtacagtg catctgtgaa gggtcgtttc
actataagca gagataattc caaaaacaca 240ctgtacctgc agatgaacag
cctgcgtgct gaggacactg ccgtctatta ttgtgctaga 300gataactggt
ttgcttactg gggccaaggg actctggtca ccgtctcctc ggctagcacc
360aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 420gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 540tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc
acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt
660gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1260aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1320ctctccctgt ctccgggtaa atgactcgag
135050669DNAArtificial SequenceSynthetic (nucleotide sequence of
H1-light) 50gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga
gagggccacc 60atcaactgca agtccagcca gagtctttta gctagcggca accaaaataa
ctacttagct 120tggcaccagc agaaaccagg acagcctcct aagatgctca
ttatttgggc
atctacccgg 180gtatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca
gtttattact gtcagcaatc ctatagtgct 300cctctcacgt tcggaggcgg
taccaaggtg gagatcaaac gtacggtggc tgcaccatct 360gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc
420ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga
taacgccctc 480caatcgggta actcccagga gagtgtcaca gagcaggaca
gcaaggacag cacctacagc 540ctcagcagca ccctgacgct gagcaaagca
gactacgaga aacacaaagt ctacgcctgc 600gaagtcaccc atcagggcct
gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660tgactcgag
66951669DNAArtificial SequenceSynthetic (nucleotide sequence of
H2-light) 51gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga
gccggcctcc 60atctcctgca agtccagtca gagtctttta gctagtggca accaaaataa
ctacttggcc 120tggcacctgc agaagccagg gcagtctcca cagatgctga
tcatttgggc atccactagg 180gtatctggag tcccagacag gttcagtggc
agtgggtcag gcactgattt cacactgaaa 240atcagcaggg tggaggctga
ggatgttgga gtttattact gccagcagtc ctacagcgct 300ccgctcacgt
tcggacaggg taccaagctg gagctcaaac gtacggtggc tgcaccatct
360gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 420ctgctgaata acttctatcc cagagaggcc aaagtacagt
ggaaggtgga taacgccctc 480caatcgggta actcccagga gagtgtcaca
gagcaggaca gcaaggacag cacctacagc 540ctcagcagca ccctgacgct
gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600gaagtcaccc
atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660tgactcgag
66952669DNAArtificial SequenceSynthetic (nucleotide sequence of
H3-light) 52gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga
gagggccacc 60atcaactgca agtccagcca gagtctttta gctagcggca accaaaataa
ctacttagct 120tggtaccagc agaaaccagg acagcctcct aagctgctca
ttatttgggc atctacccgg 180gtatccgggg tccctgaccg attcagtggc
agcgggtctg ggacagattt cactctcacc 240atcagcagcc tgcaggctga
agatgtggca gtttattact gtcagcaatc ctatagtgct 300cctctcacgt
tcggaggcgg taccaaggtg gagatcaaac gtacggtggc tgcaccatct
360gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 420ctgctgaata acttctatcc cagagaggcc aaagtacagt
ggaaggtgga taacgccctc 480caatcgggta actcccagga gagtgtcaca
gagcaggaca gcaaggacag cacctacagc 540ctcagcagca ccctgacgct
gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600gaagtcaccc
atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660tgactcgag
66953669DNAArtificial SequenceSynthetic (nucleotide sequence of
H4-light) 53gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga
tagggtcacc 60atcacctgca agtccagtca gagtctttta gctagtggca accaaaataa
ctacttggcc 120tggcaccaac agaaaccagg aaaagctccg aaaatgctga
ttatttgggc atccactagg 180gtatctggag tcccttctcg cttctctgga
tccgggtctg ggacggattt cactctgacc 240atcagcagtc tgcagccgga
agacttcgca acttattact gtcagcagtc ctacagcgct 300ccgctcacgt
tcggacaggg taccaaggtg gagatcaaac gtacggtggc tgcaccatct
360gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 420ctgctgaata acttctatcc cagagaggcc aaagtacagt
ggaaggtgga taacgccctc 480caatcgggta actcccagga gagtgtcaca
gagcaggaca gcaaggacag cacctacagc 540ctcagcagca ccctgacgct
gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600gaagtcaccc
atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660tgactcgag
6695423PRTArtificial SequenceSynthetic (linker between VH and VL)
54Gly Leu Gly Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1
5 10 15 Gly Ser Ser Gly Val Gly Ser 20 551088DNAArtificial
SequenceSynthetic (polynucleotide encoding scFv of huAbF46
antibody) 55gctagcgttt tagcagaagt tcaattggtt gaatctggtg gtggtttggt
tcaaccaggt 60ggttctttga gattgtcttg tgctgcttct ggttttactt tcaccgatta
ttacatgtcc 120tgggttagac aagctccagg taaaggtttg gaatggttgg
gtttcattag aaacaaggct 180aacggttaca ctaccgaata ttctgcttct
gttaagggta gattcaccat ttctagagac 240aactctaaga acaccttgta
cttgcaaatg aactccttga gagctgaaga tactgctgtt 300tattactgcg
ctagagataa ttggtttgct tattggggtc aaggtacttt ggttactgtt
360tcttctggcc tcgggggcct cggaggagga ggtagtggcg gaggaggctc
cggtggatcc 420agcggtgtgg gttccgatat tcaaatgacc caatctccat
cttctttgtc tgcttcagtt 480ggtgatagag ttaccattac ttgtaagtcc
tcccaatctt tgttggcttc tggtaatcag 540aacaattact tggcttggca
tcaacaaaaa ccaggtaaag ctccaaagat gttgattatt 600tgggcttcta
ccagagtttc tggtgttcca tctagatttt ctggttctgg ttccggtact
660gattttactt tgaccatttc atccttgcaa ccagaagatt tcgctactta
ctactgtcaa 720caatcttact ctgctccatt gacttttggt caaggtacaa
aggtcgaaat caagagagaa 780ttcggtaagc ctatccctaa ccctctcctc
ggtctcgatt ctacgggtgg tggtggatct 840ggtggtggtg gttctggtgg
tggtggttct caggaactga caactatatg cgagcaaatc 900ccctcaccaa
ctttagaatc gacgccgtac tctttgtcaa cgactactat tttggccaac
960gggaaggcaa tgcaaggagt ttttgaatat tacaaatcag taacgtttgt
cagtaattgc 1020ggttctcacc cctcaacaac tagcaaaggc agccccataa
acacacagta tgttttttga 1080gtttaaac 1088565597DNAArtificial
SequenceSynthetic (expression vector including polynucleotide
encoding scFv of huAbF46 antibody) 56acggattaga agccgccgag
cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60cctcgtcttc accggtcgcg
ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120acaataaaga
ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
180ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat
gataatgcga 240ttagtttttt agccttattt ctggggtaat taatcagcga
agcgatgatt tttgatctat 300taacagatat ataaatgcaa aaactgcata
accactttaa ctaatacttt caacattttc 360ggtttgtatt acttcttatt
caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420ctctatactt
taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac
480gactcactat agggaatatt aagctaattc tacttcatac attttcaatt
aagatgcagt 540tacttcgctg tttttcaata ttttctgtta ttgctagcgt
tttagcagaa gttcaattgg 600ttgaatctgg tggtggtttg gttcaaccag
gtggttcttt gagattgtct tgtgctgctt 660ctggttttac tttcaccgat
tattacatgt cctgggttag acaagctcca ggtaaaggtt 720tggaatggtt
gggtttcatt agaaacaagg ctaacggtta cactaccgaa tattctgctt
780ctgttaaggg tagattcacc atttctagag acaactctaa gaacaccttg
tacttgcaaa 840tgaactcctt gagagctgaa gatactgctg tttattactg
cgctagagat aattggtttg 900cttattgggg tcaaggtact ttggttactg
tttcttctgg cctcgggggc ctcggaggag 960gaggtagtgg cggaggaggc
tccggtggat ccagcggtgt gggttccgat attcaaatga 1020cccaatctcc
atcttctttg tctgcttcag ttggtgatag agttaccatt acttgtaagt
1080cctcccaatc tttgttggct tctggtaatc agaacaatta cttggcttgg
catcaacaaa 1140aaccaggtaa agctccaaag atgttgatta tttgggcttc
taccagagtt tctggtgttc 1200catctagatt ttctggttct ggttccggta
ctgattttac tttgaccatt tcatccttgc 1260aaccagaaga tttcgctact
tactactgtc aacaatctta ctctgctcca ttgacttttg 1320gtcaaggtac
aaaggtcgaa atcaagagag aattcggtaa gcctatccct aaccctctcc
1380tcggtctcga ttctacgggt ggtggtggat ctggtggtgg tggttctggt
ggtggtggtt 1440ctcaggaact gacaactata tgcgagcaaa tcccctcacc
aactttagaa tcgacgccgt 1500actctttgtc aacgactact attttggcca
acgggaaggc aatgcaagga gtttttgaat 1560attacaaatc agtaacgttt
gtcagtaatt gcggttctca cccctcaaca actagcaaag 1620gcagccccat
aaacacacag tatgtttttt gagtttaaac ccgctgatct gataacaaca
1680gtgtagatgt aacaaaatcg actttgttcc cactgtactt ttagctcgta
caaaatacaa 1740tatacttttc atttctccgt aaacaacatg ttttcccatg
taatatcctt ttctattttt 1800cgttccgtta ccaactttac acatacttta
tatagctatt cacttctata cactaaaaaa 1860ctaagacaat tttaattttg
ctgcctgcca tatttcaatt tgttataaat tcctataatt 1920tatcctatta
gtagctaaaa aaagatgaat gtgaatcgaa tcctaagaga attgggcaag
1980tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta
gcatttttga 2040cgaaatttgc tattttgtta gagtctttta caccatttgt
ctccacacct ccgcttacat 2100caacaccaat aacgccattt aatctaagcg
catcaccaac attttctggc gtcagtccac 2160cagctaacat aaaatgtaag
ctctcggggc tctcttgcct tccaacccag tcagaaatcg 2220agttccaatc
caaaagttca cctgtcccac ctgcttctga atcaaacaag ggaataaacg
2280aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga
aatacgagtc 2340ttttaataac tggcaaaccg aggaactctt ggtattcttg
ccacgactca tctccgtgca 2400gttggacgat atcaatgccg taatcattga
ccagagccaa aacatcctcc ttaggttgat 2460tacgaaacac gccaaccaag
tatttcggag tgcctgaact atttttatat gcttttacaa 2520gacttgaaat
tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata
2580taatacccag caagtcagca tcggaatcta gagcacattc tgcggcctct
gtgctctgca 2640agccgcaaac tttcaccaat ggaccagaac tacctgtgaa
attaataaca gacatactcc 2700aagctgcctt tgtgtgctta atcacgtata
ctcacgtgct caatagtcac caatgccctc 2760cctcttggcc ctctcctttt
cttttttcga ccgaatttct tgaagacgaa agggcctcgt 2820gatacgccta
tttttatagg ttaatgtcat gataataatg gtttcttagg acggatcgct
2880tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg
gaatttactc 2940tgtgtttatt tatttttatg ttttgtattt ggattttaga
aagtaaataa agaaggtaga 3000agagttacgg aatgaagaaa aaaaaataaa
caaaggttta aaaaatttca acaaaaagcg 3060tactttacat atatatttat
tagacaagaa aagcagatta aatagatata cattcgatta 3120acgataagta
aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat
3180gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt
agtatttgtt 3240ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa
aactattttt tctttaattt 3300ctttttttac tttctatttt taatttatat
atttatatta aaaaatttaa attataatta 3360tttttatagc acgtgatgaa
aaggacccag gtggcacttt tcggggaaat gtgcgcggaa 3420cccctatttg
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
3480cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
catttccgtg 3540tcgcccttat tccctttttt gcggcatttt gccttcctgt
ttttgctcac ccagaaacgc 3600tggtgaaagt aaaagatgct gaagatcagt
tgggtgcacg agtgggttac atcgaactgg 3660atctcaacag cggtaagatc
cttgagagtt ttcgccccga agaacgtttt ccaatgatga 3720gcacttttaa
agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
3780aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
ccagtcacag 3840aaaagcatct tacggatggc atgacagtaa gagaattatg
cagtgctgcc ataaccatga 3900gtgataacac tgcggccaac ttacttctga
caacgatcgg aggaccgaag gagctaaccg 3960cttttttgca caacatgggg
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4020atgaagccat
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
4080tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
ttaatagact 4140ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
ggcccttccg gctggctggt 4200ttattgctga taaatctgga gccggtgagc
gtgggtctcg cggtatcatt gcagcactgg 4260ggccagatgg taagccctcc
cgtatcgtag ttatctacac gacgggcagt caggcaacta 4320tggatgaacg
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
4380tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
ttttaattta 4440aaaggatcta ggtgaagatc ctttttgata atctcatgac
caaaatccct taacgtgagt 4500tttcgttcca ctgagcgtca gaccccgtag
aaaagatcaa aggatcttct tgagatcctt 4560tttttctgcg cgtaatctgc
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 4620gtttgccgga
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
4680agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
aagaactctg 4740tagcaccgcc tacatacctc gctctgctaa tcctgttacc
agtggctgct gccagtggcg 4800ataagtcgtg tcttaccggg ttggactcaa
gacgatagtt accggataag gcgcagcggt 4860cgggctgaac ggggggttcg
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 4920tgagatacct
acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg
4980acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
cttccagggg 5040ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca
cctctgactt gagcgtcgat 5100ttttgtgatg ctcgtcaggg gggccgagcc
tatggaaaaa cgccagcaac gcggcctttt 5160tacggttcct ggccttttgc
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5220attctgtgga
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
5280cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
cgcaaaccgc 5340ctctccccgc gcgttggccg attcattaat gcagctggca
cgacaggttt cccgactgga 5400aagcgggcag tgagcgcaac gcaattaatg
tgagttacct cactcattag gcaccccagg 5460ctttacactt tatgcttccg
gctcctatgt tgtgtggaat tgtgagcgga taacaatttc 5520acacaggaaa
cagctatgac catgattacg ccaagctcgg aattaaccct cactaaaggg
5580aacaaaagct ggctagt 55975713PRTArtificial SequenceSynthetic
(U6-HC7 hinge) 57Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys
Pro 1 5 10 58435DNAArtificial SequenceSynthetic (polynucleotide
encoding CDR-L3 derived from L3-1 clone) 58gaattcacta gtgattaatt
cgccgccacc atggattcac aggcccaggt cctcatgttg 60ctgctgctat cggtatctgg
tacctgtgga gatatccaga tgacccagtc cccgagctcc 120ctgtccgcct
ctgtgggcga tagggtcacc atcacctgca agtccagtca gagtctttta
180gctagtggca accaaaataa ctacttggcc tggcaccaac agaaaccagg
aaaagctccg 240aaaatgctga ttatttgggc atccactagg gtatctggag
tcccttctcg cttctctgga 300tccgggtctg ggacggattt cactctgacc
atcagcagtc tgcagccgga agacttcgca 360acttattact gtcagcagtc
ctacagccgc ccgtacacgt tcggacaggg taccaaggtg 420gagatcaaac gtacg
43559435DNAArtificial SequenceSynthetic (polynucleotide encoding
CDR-L3 derived from L3-2 clone) 59gaattcacta gtgattaatt cgccgccacc
atggattcac aggcccaggt cctcatgttg 60ctgctgctat cggtatctgg tacctgtgga
gatatccaga tgacccagtc cccgagctcc 120ctgtccgcct ctgtgggcga
tagggtcacc atcacctgca agtccagtca gagtctttta 180gctagtggca
accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg
240aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg
cttctctgga 300tccgggtctg ggacggattt cactctgacc atcagcagtc
tgcagccgga agacttcgca 360acttattact gtgggcagtc ctacagccgt
ccgctcacgt tcggacaggg taccaaggtg 420gagatcaaac gtacg
43560435DNAArtificial SequenceSynthetic (polynucleotide encoding
CDR-L3 derived from L3-3 clone) 60gaattcacta gtgattaatt cgccgccacc
atggattcac aggcccaggt cctcatgttg 60ctgctgctat cggtatctgg tacctgtgga
gatatccaga tgacccagtc cccgagctcc 120ctgtccgcct ctgtgggcga
tagggtcacc atcacctgca agtccagtca gagtctttta 180gctagtggca
accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg
240aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg
cttctctgga 300tccgggtctg ggacggattt cactctgacc atcagcagtc
tgcagccgga agacttcgca 360acttattact gtgcacagtc ctacagccat
ccgttctctt tcggacaggg taccaaggtg 420gagatcaaac gtacg
43561435DNAArtificial SequenceSynthetic (polynucleotide encoding
CDR-L3 derived from L3-5 clone) 61gaattcacta gtgattaatt cgccgccacc
atggattcac aggcccaggt cctcatgttg 60ctgctgctat cggtatctgg tacctgtgga
gatatccaga tgacccagtc cccgagctcc 120ctgtccgcct ctgtgggcga
tagggtcacc atcacctgca agtccagtca gagtctttta 180gctagtggca
accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg
240aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg
cttctctgga 300tccgggtctg ggacggattt cactctgacc atcagcagtc
tgcagccgga agacttcgca 360acttattact gtcagcagtc ctacagccgc
ccgtttacgt tcggacaggg taccaaggtg 420gagatcaaac gtacg
43562462PRTArtificial SequenceSynthetic (polypeptide consisting of
heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge and
constant region of human IgG1) 62Met Glu Trp Ser Trp Val Phe Leu
Val Thr Leu Leu Asn Gly Ile Gln 1 5 10 15 Cys Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 20 25 30 Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp 35 40 45 Tyr Tyr
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 50 55 60
Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser 65
70 75 80 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Asn Trp Phe Ala
Tyr Trp Gly Gln Gly Thr 115 120 125 Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185
190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Cys His 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310
315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410
415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 450 455 460 631410DNAArtificial SequenceSynthetic
(polynucleotide encoding polypeptide consisting of heavy chain
variable region of huAbF46-H4-A1, U6-HC7 hinge and constant region
of human IgG1) 63gaattcgccg ccaccatgga atggagctgg gtttttctcg
taacactttt aaatggtatc 60cagtgtgagg ttcagctggt ggagtctggc ggtggcctgg
tgcagccagg gggctcactc 120cgtttgtcct gtgcagcttc tggcttcacc
ttcactgatt actacatgag ctgggtgcgt 180caggccccgg gtaagggcct
ggaatggttg ggttttatta gaaacaaagc taatggttac 240acaacagagt
acagtgcatc tgtgaagggt cgtttcacta taagcagaga taattccaaa
300aacacactgt acctgcagat gaacagcctg cgtgctgagg acactgccgt
ctattattgt 360gctagagata actggtttgc ttactggggc caagggactc
tggtcaccgt ctcctcggct 420agcaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 480acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
600ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 660atctgcaacg tgaatcacaa gcccagcaac accaaggtgg
acaagaaagt tgagcccaaa 720agctgcgatt gccactgtcc tccatgtcca
gcacctgaac tcctgggggg accgtcagtc 780ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca 840tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac
900ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
cagcacgtac 960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa ggagtacaag 1020tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc caaagccaaa 1080gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag
1200tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gctggactcc 1260gacggctcct tcttcctcta cagcaagctc accgtggaca
agagcaggtg gcagcagggg 1320aacgtcttct catgctccgt gatgcatgag
gctctgcaca accactacac gcagaagagc 1380ctctccctgt ctccgggtaa
atgactcgag 141064461PRTArtificial SequenceSynthetic (polypeptide
consisting of heavy chain variable region of huAbF46-H4-A1, human
IgG2 hinge and constant region of human IgG1) 64Met Glu Trp Ser Trp
Val Phe Leu Val Thr Leu Leu Asn Gly Ile Gln 1 5 10 15 Cys Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 20 25 30 Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp 35 40
45 Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
50 55 60 Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu
Tyr Ser 65 70 75 80 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Asn Trp
Phe Ala Tyr Trp Gly Gln Gly Thr 115 120 125 Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Lys Val Glu Arg
Lys Cys Cys Val Glu Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295
300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420
425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460 651407DNAArtificial SequenceSynthetic (polynucleotide
encoding polypeptide consisting of heavy chain variable region of
huAbF46-H4-A1, human IgG2 hinge and constant region of human IgG1)
65gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc
60cagtgtgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc
120cgtttgtcct gtgcagcttc tggcttcacc ttcactgatt actacatgag
ctgggtgcgt 180caggccccgg gtaagggcct ggaatggttg ggttttatta
gaaacaaagc taatggttac 240acaacagagt acagtgcatc tgtgaagggt
cgtttcacta taagcagaga taattccaaa 300aacacactgt acctgcagat
gaacagcctg cgtgctgagg acactgccgt ctattattgt 360gctagagata
actggtttgc ttactggggc caagggactc tggtcaccgt ctcctcggct
420agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac
ctctgggggc 480acagcggccc tgggctgcct ggtcaaggac tacttccccg
aaccggtgac ggtgtcgtgg 540aactcaggcg ccctgaccag cggcgtgcac
accttcccgg ctgtcctaca gtcctcagga 600ctctactccc tcagcagcgt
ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660atctgcaacg
tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagaggaag
720tgctgtgtgg agtgcccccc ctgcccagca cctgaactcc tggggggacc
gtcagtcttc 780ctcttccccc caaaacccaa ggacaccctc atgatctccc
ggacccctga ggtcacatgc 840gtggtggtgg acgtgagcca cgaagaccct
gaggtcaagt tcaactggta cgtggacggc 900gtggaggtgc ataatgccaa
gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960gtggtcagcg
tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc
1020aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa
agccaaaggg 1080cagccccgag aaccacaggt gtacaccctg cccccatccc
gggaggagat gaccaagaac 1140caggtcagcc tgacctgcct ggtcaaaggc
ttctatccca gcgacatcgc cgtggagtgg 1200gagagcaatg ggcagccgga
gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260ggctccttct
tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac
1320gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca
gaagagcctc 1380tccctgtctc cgggtaaatg actcgag 140766460PRTArtificial
SequenceSynthetic (polypeptide consisting of heavy chain variable
region of huAbF46-H4-A1, human IgG2 hinge and constant region of
human IgG2) 66Met Glu Trp Ser Trp Val Phe Leu Val Thr Leu Leu Asn
Gly Ile Gln 1 5 10 15 Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 20 25 30 Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Thr Asp 35 40 45 Tyr Tyr Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp 50 55 60 Leu Gly Phe Ile Arg
Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser 65 70 75 80 Ala Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110 Tyr Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr
115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205 Asn Phe Gly
Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 210 215 220 Asn
Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys 225 230
235 240 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val 260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Gln Phe 275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 290 295 300 Arg Glu Glu Gln Phe Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Val His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 325 330 335 Ser Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 340 345 350
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 355
360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly 370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 420 425 430 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 435 440 445 Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 671404DNAArtificial
SequenceSynthetic (polynucleotide encoding polypeptide consisting
of heavy chain variable region of huAbF46-H4-A1, human IgG2 hinge
and constant region of human IgG2) 67gaattcgccg ccaccatgga
atggagctgg gtttttctcg taacactttt aaatggtatc 60cagtgtgagg ttcagctggt
ggagtctggc ggtggcctgg tgcagccagg gggctcactc 120cgtttgtcct
gtgcagcttc tggcttcacc ttcactgatt actacatgag ctgggtgcgt
180caggccccgg gtaagggcct ggaatggttg ggttttatta gaaacaaagc
taatggttac 240acaacagagt acagtgcatc tgtgaagggt cgtttcacta
taagcagaga taattccaaa 300aacacactgt acctgcagat gaacagcctg
cgtgctgagg acactgccgt ctattattgt 360gctagagata actggtttgc
ttactggggc caagggactc tggtcaccgt ctcctcggct 420agcaccaagg
gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc
480acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac
ggtgtcgtgg 540aactcaggcg ctctgaccag cggcgtgcac accttcccag
ctgtcctaca gtcctcagga 600ctctactccc tcagcagcgt ggtgaccgtg
ccctccagca acttcggcac ccagacctac 660acctgcaacg tagatcacaa
gcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720tgttgtgtcg
agtgcccacc gtgcccagca ccacctgtgg caggaccgtc agtcttcctc
780ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt
cacgtgcgtg 840gtggtggacg tgagccacga agaccccgag gtccagttca
actggtacgt ggacggcgtg 900gaggtgcata atgccaagac aaagccacgg
gaggagcagt tcaacagcac gttccgtgtg 960gtcagcgtcc tcaccgttgt
gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020gtctccaaca
aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag
1080ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac
caagaaccag 1140gtcagcctga cctgcctggt caaaggcttc taccccagcg
acatcgccgt ggagtgggag 1200agcaatgggc agccggagaa caactacaag
accacgcctc ccatgctgga ctccgacggc 1260tccttcttcc tctacagcaa
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc
1380ctgtctccgg gtaaatgact cgag 140468240PRTArtificial
SequenceSynthetic (polypeptide consisting of light chain variable
region of huAbF46-H4-A1(H36Y) and human kappa constant region)
68Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Ser Val Ser 1
5 10 15 Gly Thr Cys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser
Ser Gln Ser 35 40 45 Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu
Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Lys Ala Pro Lys Met Leu
Ile Ile Trp Ala Ser Thr Arg 65 70 75 80 Val Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 100 105 110 Tyr Cys Gln
Gln Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr 115 120 125 Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135
140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240
69758DNAArtificial SequenceSynthetic (polynucleotide encoding
polypeptide consisting of light chain variable region of
huAbF46-H4-A1(H36Y) and human kappa constant region) 69aattcactag
tgattaattc gccgccacca tggattcaca ggcccaggtc ctcatgttgc 60tgctgctatc
ggtatctggt acctgtggag atatccagat gacccagtcc ccgagctccc
120tgtccgcctc tgtgggcgat agggtcacca tcacctgcaa gtccagtcag
agtcttttag 180ctagtggcaa ccaaaataac tacttggcct ggtaccaaca
gaaaccagga aaagctccga 240aaatgctgat tatttgggca tccactaggg
tatctggagt cccttctcgc ttctctggat 300ccgggtctgg gacggatttc
actctgacca tcagcagtct gcagccggaa gacttcgcaa 360cttattactg
tcagcagtcc tacagccgcc cgtacacgtt cggacagggt accaaggtgg
420agatcaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct
gatgagcagt 480tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa
cttctatccc agagaggcca 540aagtacagtg gaaggtggat aacgccctcc
aatcgggtaa ctcccaggag agtgtcacag 600agcaggacag caaggacagc
acctacagcc tcagcagcac cctgacgctg agcaaagcag 660actacgagaa
acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg
720tcacaaagag cttcaacagg ggagagtgtt gactcgag 75870240PRTArtificial
SequenceSynthetic (polypeptide consisting of light chain variable
region of huAbF46-H4-A1 and human kappa constant region) 70Met Asp
Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Ser Val Ser 1 5 10 15
Gly Thr Cys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20
25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln
Ser 35 40 45 Leu Leu Ala Ser Gly Asn Gln Asn Asn His Leu Ala Trp
Tyr Gln Gln 50 55 60 Lys Pro Gly Lys Ala Pro Lys Met Leu Ile Ile
Trp Ala Ser Thr Arg 65 70 75
80 Val Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr 100 105 110 Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Tyr Thr Phe
Gly Gln Gly Thr 115 120 125 Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200
205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235 240 7119PRTArtificial SequenceSynthetic
(epitope in SEMA domain of c-Met) 71Phe Ser Pro Gln Ile Glu Glu Pro
Ser Gln Cys Pro Asp Cys Val Val 1 5 10 15 Ser Ala Leu
7210PRTArtificial SequenceSynthetic (epitope in SEMA domain of
c-Met) 72Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro 1 5 10
735PRTArtificial SequenceSynthetic (epitope in SEMA domain of
c-Met) 73Glu Glu Pro Ser Gln 1 5 74117PRTArtificial
SequenceSynthetic (heavy chain variable region of anti-c-Met
antibody (AbF46 or huAbF46-H1) ) 74Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe
Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
75114PRTArtificial SequenceSynthetic ( light chain variable region
of anti-c-Met antibody (AbF46 or huAbF46-H1) ) 75Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30
Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg 761416DNAArtificial
SequenceSynthetic (nucleotide sequence of heavy chain of nti-c-Met
antibody (AbF46 or huAbF46-H1)) 76gaattcgccg ccaccatgga atggagctgg
gtttttctcg taacactttt aaatggtatc 60cagtgtgagg tgaagctggt ggagtctgga
ggaggcttgg tacagcctgg gggttctctg 120agactctcct gtgcaacttc
tgggttcacc ttcactgatt actacatgag ctgggtccgc 180cagcctccag
gaaaggcact tgagtggttg ggttttatta gaaacaaagc taatggttac
240acaacagagt acagtgcatc tgtgaagggt cggttcacca tctccagaga
taattcccaa 300agcatcctct atcttcaaat ggacaccctg agagctgagg
acagtgccac ttattactgt 360gcaagagata actggtttgc ttactggggc
caagggactc tggtcactgt ctctgcagct 420agcaccaagg gcccatcggt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 480acagcggccc
tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
540aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 600ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 660atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg acaagaaagt tgagcccaaa 720tcttgtgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag
840gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 900gtggacggcg tggaggtgca taatgccaag acaaagccgc
gggaggagca gtacaacagc 960acgtaccgtg tggtcagcgt cctcaccgtc
ctgcaccagg actggctgaa tggcaaggag 1020tacaagtgca aggtctccaa
caaagccctc ccagccccca tcgagaaaac catctccaaa 1080gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg
1140accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag
cgacatcgcc 1200gtggagtggg agagcaatgg gcagccggag aacaactaca
agaccacgcc tcccgtgctg 1260gactccgacg gctccttctt cctctacagc
aagctcaccg tggacaagag caggtggcag 1320caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380aagagcctct
ccctgtctcc gggtaaatga ctcgag 141677759DNAArtificial
SequenceSynthetic (nucleotide sequence of light chain of anti-c-Met
antibody (AbF46 or huAbF46-H1)) 77gaattcacta gtgattaatt cgccgccacc
atggattcac aggcccaggt cctcatgttg 60ctgctgctat cggtatctgg tacctgtgga
gacattttga tgacccagtc tccatcctcc 120ctgactgtgt cagcaggaga
gaaggtcact atgagctgca agtccagtca gagtctttta 180gctagtggca
accaaaataa ctacttggcc tggcaccagc agaaaccagg acgatctcct
240aaaatgctga taatttgggc atccactagg gtatctggag tccctgatcg
cttcataggc 300agtggatctg ggacggattt cactctgacc atcaacagtg
tgcaggctga agatctggct 360gtttattact gtcagcagtc ctacagcgct
ccgctcacgt tcggtgctgg gaccaagctg 420gagctgaaac gtacggtggc
tgcaccatct gtcttcatct tcccgccatc tgatgagcag 480ttgaaatctg
gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc
540aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga
gagtgtcaca 600gagcaggaca gcaaggacag cacctacagc ctcagcagca
ccctgacgct gagcaaagca 660gactacgaga aacacaaagt ctacgcctgc
gaagtcaccc atcagggcct gagctcgccc 720gtcacaaaga gcttcaacag
gggagagtgt tgactcgag 759784170DNAArtificial SequenceSynthetic
(polynucleotide encoding c-Met protein) 78atgaaggccc ccgctgtgct
tgcacctggc atcctcgtgc tcctgtttac cttggtgcag 60aggagcaatg gggagtgtaa
agaggcacta gcaaagtccg agatgaatgt gaatatgaag 120tatcagcttc
ccaacttcac cgcggaaaca cccatccaga atgtcattct acatgagcat
180cacattttcc ttggtgccac taactacatt tatgttttaa atgaggaaga
ccttcagaag 240gttgctgagt acaagactgg gcctgtgctg gaacacccag
attgtttccc atgtcaggac 300tgcagcagca aagccaattt atcaggaggt
gtttggaaag ataacatcaa catggctcta 360gttgtcgaca cctactatga
tgatcaactc attagctgtg gcagcgtcaa cagagggacc 420tgccagcgac
atgtctttcc ccacaatcat actgctgaca tacagtcgga ggttcactgc
480atattctccc cacagataga agagcccagc cagtgtcctg actgtgtggt
gagcgccctg 540ggagccaaag tcctttcatc tgtaaaggac cggttcatca
acttctttgt aggcaatacc 600ataaattctt cttatttccc agatcatcca
ttgcattcga tatcagtgag aaggctaaag 660gaaacgaaag atggttttat
gtttttgacg gaccagtcct acattgatgt tttacctgag 720ttcagagatt
cttaccccat taagtatgtc catgcctttg aaagcaacaa ttttatttac
780ttcttgacgg tccaaaggga aactctagat gctcagactt ttcacacaag
aataatcagg 840ttctgttcca taaactctgg attgcattcc tacatggaaa
tgcctctgga gtgtattctc 900acagaaaaga gaaaaaagag atccacaaag
aaggaagtgt ttaatatact tcaggctgcg 960tatgtcagca agcctggggc
ccagcttgct agacaaatag gagccagcct gaatgatgac 1020attcttttcg
gggtgttcgc acaaagcaag ccagattctg ccgaaccaat ggatcgatct
1080gccatgtgtg cattccctat caaatatgtc aacgacttct tcaacaagat
cgtcaacaaa 1140aacaatgtga gatgtctcca gcatttttac ggacccaatc
atgagcactg ctttaatagg 1200acacttctga gaaattcatc aggctgtgaa
gcgcgccgtg atgaatatcg aacagagttt 1260accacagctt tgcagcgcgt
tgacttattc atgggtcaat tcagcgaagt cctcttaaca 1320tctatatcca
ccttcattaa aggagacctc accatagcta atcttgggac atcagagggt
1380cgcttcatgc aggttgtggt ttctcgatca ggaccatcaa cccctcatgt
gaattttctc 1440ctggactccc atccagtgtc tccagaagtg attgtggagc
atacattaaa ccaaaatggc 1500tacacactgg ttatcactgg gaagaagatc
acgaagatcc cattgaatgg cttgggctgc 1560agacatttcc agtcctgcag
tcaatgcctc tctgccccac cctttgttca gtgtggctgg 1620tgccacgaca
aatgtgtgcg atcggaggaa tgcctgagcg ggacatggac tcaacagatc
1680tgtctgcctg caatctacaa ggttttccca aatagtgcac cccttgaagg
agggacaagg 1740ctgaccatat gtggctggga ctttggattt cggaggaata
ataaatttga tttaaagaaa 1800actagagttc tccttggaaa tgagagctgc
accttgactt taagtgagag cacgatgaat 1860acattgaaat gcacagttgg
tcctgccatg aataagcatt tcaatatgtc cataattatt 1920tcaaatggcc
acgggacaac acaatacagt acattctcct atgtggatcc tgtaataaca
1980agtatttcgc cgaaatacgg tcctatggct ggtggcactt tacttacttt
aactggaaat 2040tacctaaaca gtgggaattc tagacacatt tcaattggtg
gaaaaacatg tactttaaaa 2100agtgtgtcaa acagtattct tgaatgttat
accccagccc aaaccatttc aactgagttt 2160gctgttaaat tgaaaattga
cttagccaac cgagagacaa gcatcttcag ttaccgtgaa 2220gatcccattg
tctatgaaat tcatccaacc aaatctttta ttagtggtgg gagcacaata
2280acaggtgttg ggaaaaacct gaattcagtt agtgtcccga gaatggtcat
aaatgtgcat 2340gaagcaggaa ggaactttac agtggcatgt caacatcgct
ctaattcaga gataatctgt 2400tgtaccactc cttccctgca acagctgaat
ctgcaactcc ccctgaaaac caaagccttt 2460ttcatgttag atgggatcct
ttccaaatac tttgatctca tttatgtaca taatcctgtg 2520tttaagcctt
ttgaaaagcc agtgatgatc tcaatgggca atgaaaatgt actggaaatt
2580aagggaaatg atattgaccc tgaagcagtt aaaggtgaag tgttaaaagt
tggaaataag 2640agctgtgaga atatacactt acattctgaa gccgttttat
gcacggtccc caatgacctg 2700ctgaaattga acagcgagct aaatatagag
tggaagcaag caatttcttc aaccgtcctt 2760ggaaaagtaa tagttcaacc
agatcagaat ttcacaggat tgattgctgg tgttgtctca 2820atatcaacag
cactgttatt actacttggg tttttcctgt ggctgaaaaa gagaaagcaa
2880attaaagatc tgggcagtga attagttcgc tacgatgcaa gagtacacac
tcctcatttg 2940gataggcttg taagtgcccg aagtgtaagc ccaactacag
aaatggtttc aaatgaatct 3000gtagactacc gagctacttt tccagaagat
cagtttccta attcatctca gaacggttca 3060tgccgacaag tgcagtatcc
tctgacagac atgtccccca tcctaactag tggggactct 3120gatatatcca
gtccattact gcaaaatact gtccacattg acctcagtgc tctaaatcca
3180gagctggtcc aggcagtgca gcatgtagtg attgggccca gtagcctgat
tgtgcatttc 3240aatgaagtca taggaagagg gcattttggt tgtgtatatc
atgggacttt gttggacaat 3300gatggcaaga aaattcactg tgctgtgaaa
tccttgaaca gaatcactga cataggagaa 3360gtttcccaat ttctgaccga
gggaatcatc atgaaagatt ttagtcatcc caatgtcctc 3420tcgctcctgg
gaatctgcct gcgaagtgaa gggtctccgc tggtggtcct accatacatg
3480aaacatggag atcttcgaaa tttcattcga aatgagactc ataatccaac
tgtaaaagat 3540cttattggct ttggtcttca agtagccaaa ggcatgaaat
atcttgcaag caaaaagttt 3600gtccacagag acttggctgc aagaaactgt
atgctggatg aaaaattcac agtcaaggtt 3660gctgattttg gtcttgccag
agacatgtat gataaagaat actatagtgt acacaacaaa 3720acaggtgcaa
agctgccagt gaagtggatg gctttggaaa gtctgcaaac tcaaaagttt
3780accaccaagt cagatgtgtg gtcctttggc gtgctcctct gggagctgat
gacaagagga 3840gccccacctt atcctgacgt aaacaccttt gatataactg
tttacttgtt gcaagggaga 3900agactcctac aacccgaata ctgcccagac
cccttatatg aagtaatgct aaaatgctgg 3960caccctaaag ccgaaatgcg
cccatccttt tctgaactgg tgtcccggat atcagcgatc 4020ttctctactt
tcattgggga gcactatgtc catgtgaacg ctacttatgt gaacgtaaaa
4080tgtgtcgctc cgtatccttc tctgttgtca tcagaagata acgctgatga
tgaggtggac 4140acacgaccag cctccttctg ggagacatca
417079444PRTArtificial SequenceSynthetic (SEMA domain of c-Met)
79Leu His Glu His His Ile Phe Leu Gly Ala Thr Asn Tyr Ile Tyr Val 1
5 10 15 Leu Asn Glu Glu Asp Leu Gln Lys Val Ala Glu Tyr Lys Thr Gly
Pro 20 25 30 Val Leu Glu His Pro Asp Cys Phe Pro Cys Gln Asp Cys
Ser Ser Lys 35 40 45 Ala Asn Leu Ser Gly Gly Val Trp Lys Asp Asn
Ile Asn Met Ala Leu 50 55 60 Val Val Asp Thr Tyr Tyr Asp Asp Gln
Leu Ile Ser Cys Gly Ser Val 65 70 75 80 Asn Arg Gly Thr Cys Gln Arg
His Val Phe Pro His Asn His Thr Ala 85 90 95 Asp Ile Gln Ser Glu
Val His Cys Ile Phe Ser Pro Gln Ile Glu Glu 100 105 110 Pro Ser Gln
Cys Pro Asp Cys Val Val Ser Ala Leu Gly Ala Lys Val 115 120 125 Leu
Ser Ser Val Lys Asp Arg Phe Ile Asn Phe Phe Val Gly Asn Thr 130 135
140 Ile Asn Ser Ser Tyr Phe Pro Asp His Pro Leu His Ser Ile Ser Val
145 150 155 160 Arg Arg Leu Lys Glu Thr Lys Asp Gly Phe Met Phe Leu
Thr Asp Gln 165 170 175 Ser Tyr Ile Asp Val Leu Pro Glu Phe Arg Asp
Ser Tyr Pro Ile Lys 180 185 190 Tyr Val His Ala Phe Glu Ser Asn Asn
Phe Ile Tyr Phe Leu Thr Val 195 200 205 Gln Arg Glu Thr Leu Asp Ala
Gln Thr Phe His Thr Arg Ile Ile Arg 210 215 220 Phe Cys Ser Ile Asn
Ser Gly Leu His Ser Tyr Met Glu Met Pro Leu 225 230 235 240 Glu Cys
Ile Leu Thr Glu Lys Arg Lys Lys Arg Ser Thr Lys Lys Glu 245 250 255
Val Phe Asn Ile Leu Gln Ala Ala Tyr Val Ser Lys Pro Gly Ala Gln 260
265 270 Leu Ala Arg Gln Ile Gly Ala Ser Leu Asn Asp Asp Ile Leu Phe
Gly 275 280 285 Val Phe Ala Gln Ser Lys Pro Asp Ser Ala Glu Pro Met
Asp Arg Ser 290 295 300 Ala Met Cys Ala Phe Pro Ile Lys Tyr Val Asn
Asp Phe Phe Asn Lys 305 310 315 320 Ile Val Asn Lys Asn Asn Val Arg
Cys Leu Gln His Phe Tyr Gly Pro 325 330 335 Asn His Glu His Cys Phe
Asn Arg Thr Leu Leu Arg Asn Ser Ser Gly 340 345 350 Cys Glu Ala Arg
Arg Asp Glu Tyr Arg Thr Glu Phe Thr Thr Ala Leu 355 360 365 Gln Arg
Val Asp Leu Phe Met Gly Gln Phe Ser Glu Val Leu Leu Thr 370 375 380
Ser Ile Ser Thr Phe Ile Lys Gly Asp Leu Thr Ile Ala Asn Leu Gly 385
390 395 400 Thr Ser Glu Gly Arg Phe Met Gln Val Val Val Ser Arg Ser
Gly Pro 405 410 415 Ser Thr Pro His Val Asn Phe Leu Leu Asp Ser His
Pro Val Ser Pro 420 425 430 Glu Val Ile Val Glu His Thr Leu Asn Gln
Asn Gly 435 440 80451PRTArtificial SequenceSynthetic (PSI-IPT
domain of c-Met) 80Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys
Ile Pro Leu Asn 1 5 10 15 Gly Leu Gly Cys Arg His Phe Gln Ser Cys
Ser Gln Cys Leu Ser Ala 20 25 30 Pro Pro Phe Val Gln Cys Gly Trp
Cys His Asp Lys Cys Val Arg Ser 35 40 45 Glu Glu Cys Leu Ser Gly
Thr Trp Thr Gln Gln Ile Cys Leu Pro Ala 50 55 60 Ile Tyr Lys Val
Phe Pro Asn Ser Ala Pro Leu Glu Gly Gly Thr Arg 65 70 75 80 Leu Thr
Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg Asn Asn Lys Phe 85 90 95
Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu Ser Cys Thr Leu 100
105 110 Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys Thr Val Gly
Pro 115 120 125 Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile Ser
Asn Gly His 130 135 140 Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val
Asp Pro Val Ile Thr 145 150 155 160 Ser Ile Ser Pro Lys Tyr Gly Pro
Met Ala Gly Gly Thr Leu Leu Thr 165 170 175 Leu Thr Gly Asn Tyr Leu
Asn Ser Gly Asn Ser Arg His Ile Ser Ile 180 185 190 Gly Gly Lys Thr
Cys Thr Leu Lys Ser Val Ser Asn Ser Ile Leu Glu 195 200 205 Cys Tyr
Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe Ala Val Lys Leu 210 215 220
Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe Ser Tyr Arg Glu 225
230 235 240 Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser Phe Ile
Ser Thr 245 250 255 Trp Trp Lys Glu Pro Leu Asn Ile Val Ser Phe Leu
Phe Cys Phe Ala 260 265 270 Ser Gly Gly Ser Thr Ile Thr Gly Val Gly
Lys Asn Leu Asn Ser Val 275 280 285 Ser Val Pro Arg Met Val Ile Asn
Val His Glu Ala Gly Arg Asn Phe 290 295 300 Thr Val Ala Cys Gln His
Arg Ser Asn Ser Glu Ile Ile Cys Cys Thr 305 310 315 320 Thr Pro Ser
Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys Thr Lys
325 330 335 Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp
Leu Ile 340 345 350 Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys
Pro Val Met Ile 355 360 365 Ser Met Gly Asn Glu Asn Val Leu Glu Ile
Lys Gly Asn Asp Ile Asp 370 375 380 Pro Glu Ala Val Lys Gly Glu Val
Leu Lys Val Gly Asn Lys Ser Cys 385 390 395 400 Glu Asn Ile His Leu
His Ser Glu Ala Val Leu Cys Thr Val Pro Asn 405 410 415 Asp Leu Leu
Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys Gln Ala 420 425 430 Ile
Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp Gln Asn 435 440
445 Phe Thr Gly 450 81313PRTArtificial SequenceSynthetic (TyrKc
domain of c-Met) 81Val His Phe Asn Glu Val Ile Gly Arg Gly His Phe
Gly Cys Val Tyr 1 5 10 15 His Gly Thr Leu Leu Asp Asn Asp Gly Lys
Lys Ile His Cys Ala Val 20 25 30 Lys Ser Leu Asn Arg Ile Thr Asp
Ile Gly Glu Val Ser Gln Phe Leu 35 40 45 Thr Glu Gly Ile Ile Met
Lys Asp Phe Ser His Pro Asn Val Leu Ser 50 55 60 Leu Leu Gly Ile
Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu 65 70 75 80 Pro Tyr
Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr 85 90 95
His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val Ala 100
105 110 Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg Asp
Leu 115 120 125 Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val
Lys Val Ala 130 135 140 Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys
Glu Tyr Tyr Ser Val 145 150 155 160 His Asn Lys Thr Gly Ala Lys Leu
Pro Val Lys Trp Met Ala Leu Glu 165 170 175 Ser Leu Gln Thr Gln Lys
Phe Thr Thr Lys Ser Asp Val Trp Ser Phe 180 185 190 Gly Val Leu Leu
Trp Glu Leu Met Thr Arg Gly Ala Pro Pro Tyr Pro 195 200 205 Asp Val
Asn Thr Phe Asp Ile Thr Val Tyr Leu Leu Gln Gly Arg Arg 210 215 220
Leu Leu Gln Pro Glu Tyr Cys Pro Asp Pro Leu Tyr Glu Val Met Leu 225
230 235 240 Lys Cys Trp His Pro Lys Ala Glu Met Arg Pro Ser Phe Ser
Glu Leu 245 250 255 Val Ser Arg Ile Ser Ala Ile Phe Ser Thr Phe Ile
Gly Glu His Tyr 260 265 270 Val His Val Asn Ala Thr Tyr Val Asn Val
Lys Cys Val Ala Pro Tyr 275 280 285 Pro Ser Leu Leu Ser Ser Glu Asp
Asn Ala Asp Asp Glu Val Asp Thr 290 295 300 Arg Pro Ala Ser Phe Trp
Glu Thr Ser 305 310 821332DNAArtificial SequenceSynthetic
(polynucleotide encoding SEMA domain of c-Met) 82ctacatgagc
atcacatttt ccttggtgcc actaactaca tttatgtttt aaatgaggaa 60gaccttcaga
aggttgctga gtacaagact gggcctgtgc tggaacaccc agattgtttc
120ccatgtcagg actgcagcag caaagccaat ttatcaggag gtgtttggaa
agataacatc 180aacatggctc tagttgtcga cacctactat gatgatcaac
tcattagctg tggcagcgtc 240aacagaggga cctgccagcg acatgtcttt
ccccacaatc atactgctga catacagtcg 300gaggttcact gcatattctc
cccacagata gaagagccca gccagtgtcc tgactgtgtg 360gtgagcgccc
tgggagccaa agtcctttca tctgtaaagg accggttcat caacttcttt
420gtaggcaata ccataaattc ttcttatttc ccagatcatc cattgcattc
gatatcagtg 480agaaggctaa aggaaacgaa agatggtttt atgtttttga
cggaccagtc ctacattgat 540gttttacctg agttcagaga ttcttacccc
attaagtatg tccatgcctt tgaaagcaac 600aattttattt acttcttgac
ggtccaaagg gaaactctag atgctcagac ttttcacaca 660agaataatca
ggttctgttc cataaactct ggattgcatt cctacatgga aatgcctctg
720gagtgtattc tcacagaaaa gagaaaaaag agatccacaa agaaggaagt
gtttaatata 780cttcaggctg cgtatgtcag caagcctggg gcccagcttg
ctagacaaat aggagccagc 840ctgaatgatg acattctttt cggggtgttc
gcacaaagca agccagattc tgccgaacca 900atggatcgat ctgccatgtg
tgcattccct atcaaatatg tcaacgactt cttcaacaag 960atcgtcaaca
aaaacaatgt gagatgtctc cagcattttt acggacccaa tcatgagcac
1020tgctttaata ggacacttct gagaaattca tcaggctgtg aagcgcgccg
tgatgaatat 1080cgaacagagt ttaccacagc tttgcagcgc gttgacttat
tcatgggtca attcagcgaa 1140gtcctcttaa catctatatc caccttcatt
aaaggagacc tcaccatagc taatcttggg 1200acatcagagg gtcgcttcat
gcaggttgtg gtttctcgat caggaccatc aacccctcat 1260gtgaattttc
tcctggactc ccatccagtg tctccagaag tgattgtgga gcatacatta
1320aaccaaaatg gc 1332831299DNAArtificial SequenceSynthetic
(polynucleotide encoding PSI-IPT domain of c-Met) 83tacacactgg
ttatcactgg gaagaagatc acgaagatcc cattgaatgg cttgggctgc 60agacatttcc
agtcctgcag tcaatgcctc tctgccccac cctttgttca gtgtggctgg
120tgccacgaca aatgtgtgcg atcggaggaa tgcctgagcg ggacatggac
tcaacagatc 180tgtctgcctg caatctacaa ggttttccca aatagtgcac
cccttgaagg agggacaagg 240ctgaccatat gtggctggga ctttggattt
cggaggaata ataaatttga tttaaagaaa 300actagagttc tccttggaaa
tgagagctgc accttgactt taagtgagag cacgatgaat 360acattgaaat
gcacagttgg tcctgccatg aataagcatt tcaatatgtc cataattatt
420tcaaatggcc acgggacaac acaatacagt acattctcct atgtggatcc
tgtaataaca 480agtatttcgc cgaaatacgg tcctatggct ggtggcactt
tacttacttt aactggaaat 540tacctaaaca gtgggaattc tagacacatt
tcaattggtg gaaaaacatg tactttaaaa 600agtgtgtcaa acagtattct
tgaatgttat accccagccc aaaccatttc aactgagttt 660gctgttaaat
tgaaaattga cttagccaac cgagagacaa gcatcttcag ttaccgtgaa
720gatcccattg tctatgaaat tcatccaacc aaatctttta ttagtggtgg
gagcacaata 780acaggtgttg ggaaaaacct gaattcagtt agtgtcccga
gaatggtcat aaatgtgcat 840gaagcaggaa ggaactttac agtggcatgt
caacatcgct ctaattcaga gataatctgt 900tgtaccactc cttccctgca
acagctgaat ctgcaactcc ccctgaaaac caaagccttt 960ttcatgttag
atgggatcct ttccaaatac tttgatctca tttatgtaca taatcctgtg
1020tttaagcctt ttgaaaagcc agtgatgatc tcaatgggca atgaaaatgt
actggaaatt 1080aagggaaatg atattgaccc tgaagcagtt aaaggtgaag
tgttaaaagt tggaaataag 1140agctgtgaga atatacactt acattctgaa
gccgttttat gcacggtccc caatgacctg 1200ctgaaattga acagcgagct
aaatatagag tggaagcaag caatttcttc aaccgtcctt 1260ggaaaagtaa
tagttcaacc agatcagaat ttcacagga 129984939DNAArtificial
SequenceSynthetic (polynucleotide encoding TyrKc domain of c-Met)
84gtgcatttca atgaagtcat aggaagaggg cattttggtt gtgtatatca tgggactttg
60ttggacaatg atggcaagaa aattcactgt gctgtgaaat ccttgaacag aatcactgac
120ataggagaag tttcccaatt tctgaccgag ggaatcatca tgaaagattt
tagtcatccc 180aatgtcctct cgctcctggg aatctgcctg cgaagtgaag
ggtctccgct ggtggtccta 240ccatacatga aacatggaga tcttcgaaat
ttcattcgaa atgagactca taatccaact 300gtaaaagatc ttattggctt
tggtcttcaa gtagccaaag gcatgaaata tcttgcaagc 360aaaaagtttg
tccacagaga cttggctgca agaaactgta tgctggatga aaaattcaca
420gtcaaggttg ctgattttgg tcttgccaga gacatgtatg ataaagaata
ctatagtgta 480cacaacaaaa caggtgcaaa gctgccagtg aagtggatgg
ctttggaaag tctgcaaact 540caaaagttta ccaccaagtc agatgtgtgg
tcctttggcg tgctcctctg ggagctgatg 600acaagaggag ccccacctta
tcctgacgta aacacctttg atataactgt ttacttgttg 660caagggagaa
gactcctaca acccgaatac tgcccagacc ccttatatga agtaatgcta
720aaatgctggc accctaaagc cgaaatgcgc ccatcctttt ctgaactggt
gtcccggata 780tcagcgatct tctctacttt cattggggag cactatgtcc
atgtgaacgc tacttatgtg 840aacgtaaaat gtgtcgctcc gtatccttct
ctgttgtcat cagaagataa cgctgatgat 900gaggtggaca cacgaccagc
ctccttctgg gagacatca 9398513PRTArtificial SequenceSynthetic (heavy
chain CDR3 of anti-c-Met antibody) 85Asp Asn Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Leu Val 1 5 10 8610PRTArtificial SequenceSynthetic
(light chain CDR3 of anti-c-Met antibody) 86Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu 1 5 10 87117PRTArtificial SequenceSynthetic (heavy
chain variable region of monoclonal antibody AbF46) 87Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr
Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asp Thr Leu Arg Ala
Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe
Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115
88114PRTArtificial SequenceSynthetic (light chain variable region
of anti-c-Met antibody) 88Asp Ile Leu Met Thr Gln Ser Pro Ser Ser
Leu Thr Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys
Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr
Leu Ala Trp His Gln Gln Lys Pro Gly Arg 35 40 45 Ser Pro Lys Met
Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Asp
Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80
Ile Asn Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85
90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu 100 105 110 Lys Arg 8917PRTArtificial SequenceSynthetic (light
chain CDR3 of anti-c-Met antibody) 89Gln Gln Ser Tyr Ser Ala Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu 1 5 10 15 Glu
90117PRTArtificial SequenceSynthetic (heavy chain variable region
of AT-VH1) 90Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asn Lys Ala
Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr
Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 91117PRTArtificial SequenceSynthetic
(heavy chain variable region of AT-VH2) 91Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45
Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
92117PRTArtificial SequenceSynthetic (heavy chain variable region
of AT-VH3) 92Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asn Lys Ala
Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr
Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 93117PRTArtificial SequenceSynthetic
(heavy chain variable region of AT-VH4) 93Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45
Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
94117PRTArtificial SequenceSynthetic (heavy chain variable region
of AT-VH5) 94Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asn Lys Ala
Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr
Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 95114PRTArtificial SequenceSynthetic
(light chain variable region of anti c-Met humanized
antibody(huAbF46-H4)) 95Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser
Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu
Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met Leu
Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90
95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110 Lys Arg 96113PRTArtificial SequenceSynthetic (light
chain variable region of AT-Vk1) 96Asp Ile Leu Met Thr Gln Ser Pro
Ser Ser Leu Thr Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr
Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn
Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro
Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln 85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile 100 105 110 Lys 97113PRTArtificial
SequenceSynthetic (light chain variable region of AT-Vk2) 97Asp Ile
Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu
Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln
Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala
Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser
Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys
98113PRTArtificial SequenceSynthetic (light chain variable region
of AT-Vk3) 98Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln
Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp
His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met Leu Ile Ile
Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Ser
Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110 Lys 99113PRTArtificial SequenceSynthetic (light chain variable
region of AT-Vk4) 99Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser
Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala
Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met Leu Ile
Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95
Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100
105 110 Lys 10013PRTArtificial SequenceSynthetic (modified hinge
region(U7-HC6)) 100Glu Pro Ser Cys Asp Lys His Cys Cys Pro Pro Cys
Pro 1 5 10 10113PRTArtificial SequenceSynthetic (modified hinge
region(U6-HC7)) 101Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys
Pro 1 5 10 10212PRTArtificial SequenceSynthetic (modified hinge
region(U3-HC9)) 102Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro
1 5 10 10314PRTArtificial SequenceSynthetic (modified hinge
region(U6-HC8)) 103Glu Pro Arg Asp Cys Gly Cys Lys Pro Cys Pro Pro
Cys Pro 1 5 10 10413PRTArtificial SequenceSynthetic (modified hinge
region(U8-HC5)) 104Glu Lys Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 1 5 10 10515PRTArtificial SequenceSynthetic (human hinge
region) 105Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 1 5 10 15 10617PRTArtificial SequenceSynthetic (CDR-L1 of
antibody L3-11Y) 106Lys Ser Ser Gln Ser Leu Leu Ala Trp Gly Asn Gln
Asn Asn Tyr Leu 1 5 10 15 Ala 107114PRTArtificial SequenceSynthetic
(amino acid sequence of light chain variable region of antibody
L3-11Y) 107Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser
Leu Leu Ala Trp 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met Leu Ile Ile Trp
Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr
Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110
Lys Arg 108220PRTArtificial SequenceSynthetic (amino acid sequence
of light chain of antibody L3-11Y) 108Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Trp 20 25 30 Gly Asn Gln
Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala
Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55
60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln 85 90 95 Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
220 10920DNAArtificial SequenceSynthetic (c-Met forward primer)
109acctgccagc gacatgtctt 2011023DNAArtificial SequenceSynthetic
(c-Met reverse primer) 110gacactggct gggctcttct atc
2311125DNAArtificial SequenceSynthetic (Actin forward primer)
111tcacccacac tgtgcccatc tacga 2511223DNAArtificial
SequenceSynthetic (Actin reverse primer) 112tcggtgagga tcttcatgag
gta 231131338PRTArtificial SequenceSynthetic (human VEGFR-1) 113Met
Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10
15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro
20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly
Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys
Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu
Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe
Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr
Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys
Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp
Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145
150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu
Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser
Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile
Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr
Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile
Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu
Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro
Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265
270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His
275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln
Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly
Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr
Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val
Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met
Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys
Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr
Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390
395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe
Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln
Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu
Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr
Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys
Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser
Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met
Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510
Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515
520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr
Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn
Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly
Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr
Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg
Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr
Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val
Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635
640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg
645 650 655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His
Thr Val 660 665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala
Asn Gly Val Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn
His Lys Ile Gln Gln Glu 690 695 700 Pro Gly Ile Ile Leu Gly Pro Gly
Ser Ser Thr Leu Phe Ile Glu Arg 705 710 715 720 Val Thr Glu Glu Asp
Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln 725 730 735 Lys Gly Ser
Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser 740 745 750 Asp
Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr Cys Thr Cys Val Ala 755 760
765 Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys Met Lys
770 775 780 Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile
Met Asp 785 790 795 800 Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu
Arg Leu Pro Tyr Asp 805 810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu
Arg Leu Lys Leu Gly Lys Ser 820 825 830 Leu Gly Arg Gly Ala Phe Gly
Lys Val Val Gln Ala Ser Ala Phe Gly 835 840 845 Ile Lys Lys Ser Pro
Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys 850 855 860 Glu Gly Ala
Thr Ala Ser Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys 865 870 875 880
Ile Leu Thr His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly 885
890 895 Ala Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr
Cys 900 905 910 Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg
Asp Leu Phe 915 920 925 Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu
Pro Lys Lys Glu Lys 930 935 940 Met Glu Pro Gly Leu Glu Gln Gly Lys
Lys Pro Arg Leu Asp Ser Val 945 950 955 960 Thr Ser Ser Glu Ser Phe
Ala Ser Ser Gly Phe Gln Glu Asp Lys Ser 965 970 975 Leu Ser Asp Val
Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 980 985 990 Pro Ile
Thr Met Glu Asp Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000
1005 Gly Met Glu Phe Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu
1010 1015 1020 Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val
Lys Ile 1025 1030 1035 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys
Asn Pro Asp Tyr 1040 1045 1050 Val Arg Lys Gly Asp Thr Arg Leu Pro
Leu Lys Trp Met Ala Pro 1055 1060 1065 Glu Ser Ile Phe Asp Lys Ile
Tyr Ser Thr Lys Ser Asp Val Trp 1070 1075 1080 Ser Tyr Gly Val Leu
Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser 1085 1090 1095 Pro Tyr Pro
Gly Val Gln Met Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110 Arg
Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120
1125 Ile Tyr Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu
1130 1135 1140 Arg Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp
Leu Leu 1145 1150 1155 Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr
Ile Pro Ile Asn 1160 1165 1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe
Thr Tyr Ser Thr Pro Ala 1175 1180 1185 Phe Ser Glu Asp Phe Phe Lys
Glu Ser Ile Ser Ala Pro Lys Phe 1190 1195 1200 Asn Ser Gly Ser Ser
Asp Asp Val Arg Tyr Val Asn Ala Phe Lys 1205 1210 1215 Phe Met Ser
Leu Glu Arg Ile Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230 Asn
Ala Thr Ser Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240
1245 Leu Leu Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser
1250 1255 1260 Lys Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr
Ser Lys 1265 1270 1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg
Pro Ser Phe Cys 1280 1285 1290 His Ser Ser Cys Gly His Val Ser Glu
Gly Lys Arg Arg Phe Thr 1295 1300 1305 Tyr Asp His Ala Glu Leu Glu
Arg Lys Ile Ala Cys Cys Ser Pro 1310 1315 1320 Pro Pro Asp Tyr Asn
Ser Val Val Leu Tyr Ser Thr Pro Pro Ile 1325 1330 1335
114101PRTArtificial SequenceSynthetic
(amino acid sequence of Ig2 domain(VIG2) of human VEGF receptor 1)
114Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys
Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe
Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp
Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys
Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His
Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr
Ile 100 115303DNAArtificial SequenceSynthetic (coding sequence of
VIG2) 115agtgatacag gtagaccttt cgtagagatg tacagtgaaa tccccgaaat
tatacacatg 60actgaaggaa gggagctcgt cattccctgc cgggttacgt cacctaacat
cactgttact 120ttaaaaaagt ttccacttga cactttgatc cctgatggaa
aacgcataat ctgggacagt 180agaaagggct tcatcatatc aaatgcaacg
tacaaagaaa tagggcttct gacctgtgaa 240gcaacagtca atgggcattt
gtataagaca aactatctca cacatcgaca aaccaataca 300atc
3031165PRTArtificial SequenceSynthetic (linker (G4S) ) 116Gly Gly
Gly Gly Ser 1 5 11710PRTArtificial SequenceSynthetic (linker
(G4S)2) 117Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
11820PRTArtificial SequenceSynthetic (linker (G4S)4) 118Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly
Gly Gly Ser 20
* * * * *